Oxidized lipids and methods of use thereof

ABSTRACT

The present invention is directed to oxidized lipids and pharmaceutical compositions comprising the same. The present invention is also directed to methods of making an oxidized lipid of the invention and to methods of treating or preventing fibrosis or inflammatory diseases or disorders comprising an oxidized lipid of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/952,827, filed Nov. 25, 2015, which claims priority benefit to U.S.Provisional Appl. No. 62/085,153, filed Nov. 26, 2014, the contents ofwhich are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention, in some embodiments thereof, relates to oxidizedlipid compounds and pharmaceutical compositions comprising the same. Theinvention also relates to methods of making such compounds andcompositions, and methods of treating or preventing fibrosis orinflammatory diseases or disorders with such compounds and compositions.

BACKGROUND OF THE INVENTION

Fibrosis is the formation of excess fibrous connective tissue in anorgan or tissue, typically as the result of inflammation or damage.Fibrosis encompasses the pathological state of excess deposition offibrous tissue, as well as the process of connective tissue depositionin healing. Fibrosis is similar to the process of scarring, in that bothinvolve stimulated cells (e.g., fibroblasts) laying down connectivetissue, including collagen and glycosaminoglycans.

Fibrosis can be considered as a scarring process in response to chronicdiseases where excessive extracellular matrix (ECM) deposition leads toirreversible tissue damage and failure or disturbance of proper organfunction. The pathophysiology of fibrosis has generally been studied inthe context of the particular organ or tissue affected, including lung,kidney, liver, heart and skin. Loss of metabolic homeostasis and chroniclow-grade inflammation may play a role in the pathogenesis of fibrosis.Fibrogenesis is a dynamic process and occurs in four phases: i)initiation, due to injury of the organ/tissue; ii) inflammation andactivation of effector cells; iii) enhanced synthesis of ECM; and iv)deposition of ECM with progression to end-organ failure.

Fibrosis can occur in many tissues within the body. Examples includepulmonary fibrosis (lungs), idiopathic pulmonary fibrosis (lungs),cystic fibrosis (lungs), progressive massive fibrosis (lungs), liverfibrosis, cirrhosis (liver), steatohepatitis (fatty liver disease),nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis(NASH), endomyocardial fibrosis (heart), myocardial infarction (heart),atrial fibrosis (heart), medastinal fibrosis (soft tissue ofmediastinum), myelofibrosis (bone marrow), retroperitoneal fibrosis(soft tissue of the retroperitoneum), nephrogenic systemic fibrosis(skin), keloid (skin), Crohn's disease (intestine), scleroderma/systemicsclerosis (skin, lungs), arthrofibrosis (knee, shoulder, other joints),Peyronie's disease (penis), Dupuytren's contracture (hands, fingers),adhesive capsulitis (shoulder), kidney fibrosis, and focal and segmentalglomerulosclerosis (kidney).

One of the major complications of insulin resistance and metabolicsyndrome is nonalcoholic fatty liver disease (NAFLD), which can progressfrom fatty liver to liver inflammation (NASH) and liver fibrosis. It isbelieved that due to intestinal barrier leakage, accompanied byovergrowth and changes in the composition of gut flora, bacterialcomponents travel through the portal vein into the liver, where theyencounter toll like receptors (TLRs).

TLRs are a family of receptors imperative for the innate immune responseagainst microbial invasion. TLRs can be divided into two major subgroupsbased on their cellular localization. Plasma membrane expressed TLRsinclude TLR1, TLR2, TLR4, TLR5, and TLR6, whereas the intracellular TLRsinclude TLR3, TLR7, TLR8, and TLR9. The interaction between TLRs withtheir cognate agonists instigates a cascade of cues which includerecruitment of the adaptor molecules MyD88/TRIF and downstreamphosphorylation of MAPK kinases and NF-κB. These events culminate in thesecretion of proinflammatory cytokines, including IL-12/23, IL-6 andTNF-α. TLR2 forms a heterodimer with TLR1 which recognizes bacterialtriacylated lipopeptides, and a heterodimer with TLR6 which recognizesbacterial diacylated lipopeptides. TLR4 coupled to MD2 in complex withlipopolysaccharide-binding protein (LBP) and the co-receptor CD14 bindlipopolysaccharide (LPS) from gram negative bacteria.

Liver resident kupffer and hepatic stellate cells (HSC) express TLR2which recognize triacylated lipopeptides from Gram-negative bacteria andmycoplasma and diacylated lipopeptides from Gram-negative bacteria andmycoplasma and TLR4 and its co-receptor CD14 which recognizelipopolysaccharide (LPS) from gram-negative bacteria. Both TLR2 and TLR4can also bind to danger associated molecular patterns released frominjured tissues. These TLR2 and TLR4 complexes mediate the production ofpro-inflammatory cytokines and fibrogenic response by kupffer andstellate cells. Pre-clinical studies showed that nonalcoholicsteatohepatitis and liver fibrosis are inhibited in TLR2 and TLR4deficient mice, indicating its role in disease pathogenesis. In humans,LPS plasma levels are elevated in NAFLD patients and alterations in TLR4and CD14 genes are associated with risks of developing nonalcoholicsteatohepatitis and fibrogenesis.

Monocytes are key players in the immune system, with critical roles ininnate and adaptive immunity, immune surveillance and particlescavenging. Whereas a subset of monocytes is “resident” and recruited totissues independently of inflammatory stimuli to assist in steady-statesurveillance, wound-healing and resolution of inflammation, the absolutemajority (80-90%) of human circulating monocytes is classified as“inflammatory”. These monocytes can sense inflammatory stimuli andquickly migrate through the vascular or lymphatic endothelium to theperiphery, where they can differentiate into macrophages and dendriticcells (DCs) which cooperate with additional cell subsets (such asTh1-cells) to promote inflammation. While playing a necessary role inhost defense, monocytes were nonetheless identified as criticalmediators of several inflammatory diseases, including atherosclerosis,rheumatoid arthritis (RA) and multiple sclerosis (MS). Suppressing theaccumulation of unwanted monocytes/macrophages in a chronically inflamedtissue has therapeutic potential, and migration inhibitors haveaccordingly demonstrated promising anti-inflammatory results in animalmodels and clinical trials.

Renal fibrosis (kidney fibrosis) is a wound healing/scarring responsefollowing kidney injury that occurs in many forms of chronic kidneydisease (CKD). Following kidney injury, resident fibroblasts areactivated by various pro-inflammatory and pro-fibrotic stimuli.Activated fibroblasts, also called myofibroblasts, produce excessive ECMproteins that accumulate in the interstitium, and therefore areconsidered a mediator of renal fibrosis. Regardless of the primaryinsult leading to renal fibrosis, chronic inflammation appears to be aprocess heralding renal fibrogenesis. Elevated levels of inflammatorymarkers were associated with an increased risk of developing CKD.Induction of various pro-inflammatory cytokines interleukin (IL)-6,IL-8, IL-10, chemokine (C—C motif) ligand 2 (CCL2), tumor necrosisfactor-α (TNF-α) and adhesion molecules (intercellular adhesionmolecule-1 and vascular cell adhesion molecule-1) attracted thetransmigration of macrophages and T cells from the circulation to theinterstitium, thereby further enhancing the inflammatory state. Evidencesuggests that TLRs and macrophages are associated with the pathogenesisof renal fibrosis.

Fibrosis or inflammatory diseases or disorders can cause severemorbidity and deleterious effects on patients' daily function, activityof daily living (ADL) and quality of life, and can lead to a poorprognosis. For example, idiopathic pulmonary fibrosis (IPF) is a chronicintractable disease associated with worsening and debilitating shortnessof breath. IPF patients become oxygen dependent, and have an averagemedian survival time of three years and a five year survival rate of 20%to 40% after diagnosis. Therefore, the development of new therapies forfibrosis and inflammatory diseases or disorders is needed.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides oxidized lipidcompounds according to Formula 1,

or a stereoisomer, a stereoisomeric mixture, or a salt thereof,

wherein each of B₁, B₂, and B₃ is independently selected from the groupconsisting of oxygen, sulfur, nitrogen, phosphorus and silicon, whereineach of said nitrogen, phosphorus and silicon is optionally substitutedby one or more substituents selected from the group consisting of alkyl,halo, cycloalkyl, aryl, hydroxy, thiohydroxy, alkoxy, aryloxy,thioaryloxy, thioalkoxy, and oxo;

wherein R¹⁰ is a C₂₋₂₈ alkyl optionally substituted by one to five R¹¹substituents, wherein each R¹¹ is independently selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, phosphonate, phosphate, phosphinyl, sulfonyl, sulfinyl,sulfonamide, amide, carbonyl, thiocarbonyl, C-carboxy, O-carboxy,C-carbamate, N-carbamate, C-thiocarboxy, S-thiocarboxy, and amino;

wherein p is an integer selected from 1-10;

wherein q is an integer selected from 1-26;

wherein R²⁰ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; and

wherein Het is a heteroalicyclic ring or a heteroaryl.

Suitable B₁, B₂, and B₃ for Formula 1 are defined herein. In someembodiments, B₁ is O. In some embodiments, B₂ is O. In some embodiments,B₃ is O. In some embodiments, at least two of B₁, B₂, and B₃ are O,e.g., B₁, B₂ are O and B₃ is O or S; B₁, B₃ are O and B₂ is O or S; orB₂, B₃ are O and B₁ is O or S. In some embodiments, B₁ is S. In someembodiments, B₂ is S. In some embodiments, B₃ is S. In some embodiments,at least two of B₁, B₂, and B₃ are S, e.g., B₁, B₂ are S and B₃ is O orS; B₁, B₃ are S and B₂ is O or S; or B₂, B₃ are S and B₁ is O or S. Insome embodiments, all of B₁, B₂, and B₃ are O. In some embodiments, allof B₁, B₂, and B₃ are S.

Suitable R¹⁰ for Formula 1 are defined herein. In some embodiments, R¹⁰is a C₂₋₂₈ alkyl. In some embodiments, R¹⁰ is a straight chain C₂₋₂₈alkyl, e.g., an alkyl chain having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbons,substituted or unsubstituted. In some embodiments, R¹⁰ is a straightchain C₂₋₂₈ alkyl, e.g., an alkyl chain having 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28carbons, substituted by one to five R¹¹ substituents, wherein each R¹¹is independently as defined herein, e.g., a halogen (e.g., F) or analkyl (e.g., a C₁₋₁₀ alkyl). In some embodiments, R¹⁰ is selected fromthe group consisting of hexadecyl, dodecyl, octadecyl, octyl, eicosanyl,cis-9-hexadecenyl, (2′-octyl)dodecyl, and (15′-carboxy)pentadecyl. Insome embodiments, R¹⁰ is hexadecyl. In some embodiments, R¹⁰ is(2′-octyl)dodecyl. In some embodiments, R¹⁰ is eicosanyl.

Suitable R²⁰ for Formula 1 are defined herein. In some embodiments, R²⁰is a hydrogen or an alkyl. In some embodiments, R²⁰ is hydrogen. In someembodiments, R²⁰ is an alkyl, e.g., a C₁₋₄ alkyl (e.g., methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, or tert-butyl). Insome embodiments, R²⁰ is methyl.

Suitable values for p and q in Formula 1 are defined herein. In someembodiments, q is an integer of 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10. In some embodiments, q is 4. In some embodiments, p is an integerof 1-7, e.g., 1, 2, 3, 4, 5, 6, or 7. In some embodiments, p is 2.

Suitable Het for Formula 1 are defined herein. In some embodiments, Hetis a heteroaryl. In some embodiments, Het is a monocyclic heteroaryl. Insome embodiments, Het is a nitrogen containing heteroaryl (e.g.,monocyclic heteroaryl). In some embodiments, Het is a monocyclicheteroaryl containing 1, 2, 3, or 4 nitrogen atoms. In some embodiments,Het is a 6-member ring monocyclic heteroaryl, e.g., pyridine,pyrimidine, pyridazine, pyrazine, triazine, etc. In some embodiments,Het is a 5-member ring monocyclic heteroaryl, e.g., imidazole, thiazole,isothiazole, oxazole, isoxazole, oxidiazole, pyrazole, triazole, etc. Insome embodiments, Het is a bicyclic heteroaryl containing, e.g., 1-3nitrogen atoms, e.g., quinoline, isoquinoline, quinazoline,thienopyridine, thienopyrimidine, pyrrolopyridine, imidazopyridine, etc.In any of the embodiments described herein, Het can be a nitrogencontaining heteroaryl, wherein a nitrogen atom of the heteroaryl isdirectly connected to the alkylene chain, i.e., —(CH₂)_(p)— in Formula 1to form a cation. In some embodiments, Het is pyridine, wherein thenitrogen atom of the pyridine is directly connected to the alkylenechain, i.e., —(CH₂)_(p)— in Formula 1 to form a pyridinium salt (e.g.,an internal salt or an external salt as described herein). In someembodiments, Het is an unsubstituted pyridine, wherein the nitrogen atomof the pyridine is directly connected to the alkylene chain, i.e.,—(CH₂)_(p)— in Formula 1. In some embodiments, Het is a substitutedpyridine, wherein the nitrogen atom of the pyridine is directlyconnected to the alkylene chain, i.e., —(CH₂)_(p)— in Formula 1, whereinthe pyridine is substituted by one to five (e.g., 1, 2, 3, 4, or 5) R¹²substituents, wherein each R¹² is independently as defined herein, e.g.,a halogen (e.g., F, Cl), a C₆₋₁₀ aryl (e.g., phenyl), a heteroaryl, oran alkyl (e.g., a C₁₋₁₀ alkyl, e.g., a C₁₋₄ alkyl (e.g., methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl). In someembodiments, Het is a substituted pyridine and the pyridine issubstituted by one R¹² substituent at the 2-, 3-, or 4-position of thepyridine, wherein R¹² is as defined herein, for example, a halogen(e.g., F, Cl), a phenyl, or a methyl. In some embodiments, Het is3-fluoro-pyridine or 3-phenyl-pyridine.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 2:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof. SuitableR¹⁰, R²⁰, p, q, and Het are those as defined herein for Formula 1.

In some embodiments according to Formula 2, R¹⁰ is selected from thegroup consisting of hexadecyl, dodecyl, octadecyl, octyl, eicosanyl,cis-9-hexadecenyl, (2′-octyl)dodecyl, and (15′-carboxy)pentadecyl; R²⁰is hydrogen or an alkyl, e.g., a C₁₋₄ alkyl (e.g., methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, or tert-butyl); q isan integer of 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is aninteger of 1-7, e.g., 1, 2, 3, 4, 5, 6, or 7; and Het is anunsubstituted pyridine, wherein the nitrogen atom of the pyridine isdirectly connected to the alkylene chain, i.e., —(CH₂)_(p)— in Formula1, or Het is a substituted pyridine, wherein the nitrogen atom of thepyridine is directly connected to the alkylene chain, i.e., —(CH₂)_(p)—in Formula 1, wherein the pyridine is substituted by one to five (e.g.,1, 2, 3, 4, or 5) R¹² substituents, wherein each R¹² is independently asdefined herein, e.g., a halogen (e.g., F, Cl), a C₆₋₁₀ aryl (e.g.,phenyl), a heteroaryl (e.g., monocyclic heteroaryl), or an alkyl (e.g.,a C₁₋₁₀ alkyl, e.g., a C₁₋₄ alkyl (e.g., methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl). In someembodiments, Het is a substituted pyridine and the pyridine issubstituted by one R¹² substituent at the 2-, 3-, or 4-position of thepyridine, wherein R¹² is as defined herein, for example, a halogen(e.g., F, Cl), a phenyl, or a methyl. In some embodiments, Het is3-fluoro-pyridine or 3-phenyl-pyridine.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 3:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof, whereinR¹⁰, R¹¹, R¹², R²⁰, p, and q are as defined herein for Formula 1.

In some embodiments according to Formula 3, R¹⁰ is selected from thegroup consisting of hexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ ishydrogen or a C₁₋₄ alkyl (e.g., methyl, ethyl, propyl, isopropyl,n-butyl, sec-butyl, iso-butyl, or tert-butyl); q is an integer of 2-6,e.g., 2, 3, 4, 5, or 6; p is an integer of 2-5, e.g., 2, 3, 4, or 5; andthe pyridine is substituted by 0 to 3 (e.g., 0, 1, 2, or 3) R¹²substituents, wherein each R¹² is independently as defined herein, e.g.,a halogen (e.g., F, Cl), a C₆₋₁₀ aryl (e.g., phenyl), a heteroaryl, or aC₁₋₄ alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, tert-butyl). In some embodiments, R¹⁰ is selected from thegroup consisting of hexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ ishydrogen or methyl; q is 2, 3, 4, 5, or 6; p is 2, 3, 4, or 5; and thepyridine is substituted by 0 to 3 (e.g., 0, 1, 2, or 3) R¹²substituents, wherein each R¹² is independently a halogen (e.g., F, Cl),a C₆₋₁₀ aryl (e.g., phenyl), a heteroaryl, or a C₁₋₄ alkyl (e.g.,methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl,tert-butyl). In some embodiments, R¹⁰ is selected from the groupconsisting of hexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ ishydrogen or methyl; q is 4; p is 2; and the pyridine is substituted by0, 1, or 2 R¹² substituents, wherein each R¹² is independently a halogen(e.g., F, Cl), a phenyl, or a methyl. In some embodiments, the pyridineis substituted by one R¹¹ substituent at the 2-, 3-, or 4-position ofthe pyridine, wherein R¹¹ is as defined herein, for example, a halogen(e.g., F, Cl), a phenyl, or a methyl. In some embodiments, the pyridinering is substituted by one R¹² substituent, wherein the one R¹²substituent is fluorine or phenyl. In some embodiments, the pyridinering is 3-fluoro-pyridine or 3-phenyl-pyridine.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 4:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof. SuitableR¹⁰, R²⁰, and q are those as defined herein for Formula 1.

In some embodiments according to Formula 4, R¹⁰ is selected from thegroup consisting of hexadecyl, dodecyl, octadecyl, octyl, eicosanyl,cis-9-hexadecenyl, (2′-octyl)dodecyl, and (15′-carboxy)pentadecyl. Insome embodiments, R¹⁰ is hexadecyl. In some embodiments, R¹⁰ is(2′-octyl)dodecyl. In some embodiments, R¹⁰ is eicosanyl.

In some embodiments, R²⁰ is a hydrogen or an alkyl. In some embodiments,R²⁰ is hydrogen. In some embodiments, R²⁰ is an alkyl, e.g., a C₁₋₄alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, or tert-butyl). In some embodiments, R²⁰ is methyl.

In some embodiments, q is an integer of 1-10, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10. In some embodiments, q is 4.

In some embodiments, R¹⁰ is selected from the group consisting ofhexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ is hydrogen or a C₁₋₄alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, or tert-butyl); and q is an integer of 2-6, e.g., 2, 3, 4, 5,or 6. In some embodiments, R¹⁰ is selected from the group consisting ofhexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ is hydrogen; and q is aninteger of 2-6, e.g., 2, 3, 4, 5, or 6. In some embodiments, R¹⁰ isselected from the group consisting of hexadecyl, eicosanyl and(2′-octyl)dodecyl; R²⁰ is methyl; and q is an integer of 2-6, e.g., 2,3, 4, 5, or 6.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 5:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof, whereinR¹⁰ and R²⁰ are as defined herein for Formula 1.

In some embodiments according to Formula 5, R¹⁰ is selected from thegroup consisting of hexadecyl, dodecyl, octadecyl, octyl, eicosanyl,cis-9-hexadecenyl, (2′-octyl)dodecyl, and (15′-carboxy)pentadecyl. Insome embodiments, R¹⁰ is hexadecyl. In some embodiments, R¹⁰ is(2′-octyl)dodecyl. In some embodiments, R¹⁰ is eicosanyl.

In some embodiments, R²⁰ is a hydrogen or an alkyl. In some embodiments,R²⁰ is hydrogen. In some embodiments, R²⁰ is an alkyl, e.g., a C₁₋₄alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, or tert-butyl). In some embodiments, R²⁰ is methyl.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure of:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof.

In some embodiments, the invention provides a compound selected from thegroup consisting of(R)-1-hexadecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-701);(R)-1-eicosanyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-702);(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-703);(R)-1-hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-704); and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoricacid pyridiniumethyl ester (VB-705). The prefix “(R)-” refers to theconfiguration of the C-2 carbon of the glycerol backbone.

In some embodiments, the invention provides an oxidized lipid having astructure of:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof.

In some embodiments, the invention provides a compound selected from thegroup consisting of(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-fluoro-pyridiniumethyl ester (VB-706) and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-phenyl-pyridiniumethyl ester (VB-707). The prefix “(R)-” refers to theconfiguration of the C-2 carbon of the glycerol backbone.

In still other embodiments, the present invention providespharmaceutical compositions comprising an oxidized lipid of theinvention, methods of making an oxidized lipid of the invention, andmethods of preventing or treating fibrosis or an inflammatory disease ordisorder with an oxidized lipid or composition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention.

FIG. 1A shows the structures of the VB-701, VB-702, VB-703, VB-704 andVB-705 oxidized lipid compounds. FIG. 1B shows the structures of theVB-706 and VB-707 oxidized lipid compounds.

FIG. 2 shows VB-701 inhibits lipopolysaccharide (LPS) (TLR4)-inducedsignaling in human monocytes (primary CD14+).

FIG. 3 shows VB-701 inhibits PGN (TLR2)-induced signaling in humanmonocytes (THP-1 cell line).

FIG. 4 shows VB-701 inhibits MCP-1-induced signaling in human monocytes(THP-1 cell line).

FIG. 5 shows VB-701 inhibits chemokine-induced migration of humanmonocytes (primary CD14+).

FIG. 6 shows VB-702 inhibits LPS (TLR4)-induced signaling in humanmonocytes (primary CD14+).

FIG. 7 shows VB-702 inhibits RANTES-induced signaling in human monocytes(primary CD14+).

FIG. 8 shows VB-702 inhibits chemokine-induced migration of humanmonocytes (primary CD14+).

FIGS. 9A-9B show VB-701 and VB-702 inhibit IL-12p40 levels in humanmonocytes (primary CD14+) that are LPS (TLR4)-stimulated (FIG. 9A) andPam3CSK4 (TLR2)-stimulated (FIG. 9B).

FIGS. 10A-10B show the effect of VB-703 and VB-201 on LPS-inducedsignaling in human monocytes (primary CD14+) and LPS binding inhibitionassay. Human primary monocytes were pretreated with VB-201 or VB-703 atthe indicated concentrations followed by activation with LPS. Sampleswere analyzed by western blotting for inhibition of phosphorylation(FIG. 10A). Heat shock protein HSP90 was used for loading control. FIG.10B shows results from samples incubated with VB-201 or VB-703 at theindicated concentrations for 20 minutes before biotin—LPS (100 ng/ml)was added for an additional 15 minutes. Results are the meanfluorescence intensity (MFI) of triplicates.

FIGS. 11A-11B show the effect of VB-703 on liver fibrosis. NASH wasinduced by injection of mice with 200 μg streptozotocin (STZ) two daysafter birth and by feeding a high fat diet (HFD) from 4 weeks of age.Mice were then either treated with vehicle (negative control), VB-703 (4mg/kg), or telmisartan (10 mg/kg; positive control) at six weeks of agefor three weeks. Normal mice (not NASH-induced) were also used as acontrol. Mice were sacrificed at nine weeks of age. Staining of liverhistological samples with Sirius red was used to determine the extent offibrosis. FIG. 11A shows the mean fibrosis area following treatment (%from analyzed liver section; Mean±S.E; Normal—n=5, Vehicle—n=8,Telmisartan—n=6). FIG. 11B shows representative Sirius red staining ofliver samples following treatment (200× magnification).

FIGS. 12A-12B show the effect of VB-703 on liver inflammation. Mice weretreated and evaluated as explained in the examples. FIG. 12A shows themean liver inflammation score following treatment (Mean±S.E; Normal—n=5,Vehicle—n=8, VB—703—n=8, Telmisartan—n=6). FIG. 12B shows representativeH&E staining of liver samples following treatment (200× magnification).

FIG. 13 shows VB-703 inhibits PGN (TLR2)-induced signaling in humanmonocytes (THP-1 cell line).

FIG. 14 shows VB-703 inhibits IL-6 secretion in LPS (TLR4)-inducedsignaling in monocyte derived dendritic cells.

FIG. 15 shows VB-703 inhibits IL-12p40 secretion in LPS (TLR4)-inducedsignaling in monocyte derived dendritic cells.

FIG. 16 shows VB-704 does not inhibit LPS-biotin binding to humanmonocytes (primary CD14+).

FIG. 17 shows VB-704 inhibits chemokine-induced cell migration of humanmonocytes (primary CD14+).

FIG. 18 shows VB-705 and VB-201 inhibit TLR4-induced signaling in humanmonocytes (primary CD14+).

FIG. 19 shows VB-703 and VB-705 inhibit LPS binding in human monocytes(primary CD14+).

FIG. 20 shows VB-705 does not inhibit SDF1-induced cell migration inhuman monocytes (THP-1 cell line).

FIG. 21A-21C present bar graphs showing the expression levels of twopro-inflammatory cytokines, IL-12/23p40 (FIG. 21A) and IL-1β (FIG. 21C),and the chemokine, MCP-1 (FIG. 21B), in livers taken from NASH-inducedmice. These data show that the expression of IL-12/23p40, MCP-1, andIL-1β were significantly inhibited in livers taken from NASH-inducedmice that were treated with VB-703.

FIG. 22 presents bar graphs showing the effect of VB-703 onalbumin/creatinine (mg/mg/day). Albumin/Creatinine/Day in healthy rats(n=3) (white bar), sham operated rats (n=3) (white bar with stripes),nephrectomized rats treated with solvent control (0.5% ethanol/PBS)(black bar) (n=7), nephrectomized rats VB-703 4 mg/kg treated (n=7)(light gray bar) or nephrectomized rats telmisartan 10 mg/kg treated(n=8) (dark gray bar) were evaluated at 8 weeks. Statistical data vs.nephrectomized rats treated with solvent control (0.5% ethanol/PBS) ispresented as follows: * represents p=0.002; and ** represents p≤0.001.Abbreviations are: Nx, nephrectomized; Eth, ethanol.

FIG. 23 presents bar graphs showing the effect of VB-703 in reducing thenumber of damaged glomeruli (%). Damaged glomeruli (%) in healthy rats(n=3) (white bar), sham operated rats (n=3) (white bar with stripes),nephrectomized rats treated with solvent control (0.5% ethanol/PBS)(black bar) (n=7), nephrectomized rats VB-703 4 mg/kg treated (n=7)(light gray bar) or nephrectomized rats telmisartan 10 mg/kg treated(n=8) (dark gray bar) were evaluated at 8 weeks. Statistical data vs.nephrectomized rats treated with solvent control (0.5% ethanol/PBS) ispresented as follows: * represents p≤0.005; and ** represents p≤0.001.Abbreviations are: Nx, nephrectomized; Eth, ethanol.

FIG. 24 presents bar graphs showing the effect of VB-703 in reducingglomerular sclerosis (%). Glomerular sclerosis (%) in healthy rats (n=3)(white bar), sham operated rats (n=3) (white bar with stripes),nephrectomized rats treated with solvent control (0.5% ethanol/PBS)(black bar) (n=7), nephrectomized rats VB-703 4 mg/kg treated (n=7)(light gray bar) or nephrectomized rats telmisartan 10 mg/kg treated(n=8) (dark gray bar) were evaluated at 8 weeks. Statistical data vs.nephrectomized rats treated with solvent control (0.5% ethanol/PBS) ispresented as follows: * represents p≤0.005; and ** represents p≤0.001.Abbreviations are: Nx, nephrectomized; Eth, ethanol.

FIG. 25 presents PAS staining images (×400) showing the effect of VB-703in reducing glomerular sclerosis. Renal morphology was assessed by lightmicroscope in PAS stained sections of healthy rats (Healthy ×400), shamoperated rats (Sham ×400), nephrectomized rats treated with solventcontrol (0.5% ethanol/PBS) (Nx PBS 0.5% Eth ×400), nephrectomized ratsVB-703 4mg/kg treated (Nx VB-703 4mg/kg ×400) or nephrectomized ratstelmisartan 10 mg/kg treated (Nx Telmisartan 10 mg/kg ×400) at 8 weeksfollowing the first surgery. Abbreviations are: Nx, nephrectomized; Eth,ethanol, PAS, Periodic Acid-Schiff.

FIGS. 26A-26C present bar graphs showing the effect of VB-703 onpro-fibrotic markers. Collagen IV (FIG. 26A), fibronectin (FIG. 26B) andTGF-β (FIG. 26C) related expression in the kidney were evaluated inhealthy rats (white bar), sham operated rats (white bar with stripes),nephrectomized rats treated with solvent control (0.5% ethanol/PBS)(black bar), nephrectomized rats VB-703 4 mg/kg treated (light graybar), or nephrectomized rats telmisartan 10 mg/kg treated (dark graybar) at 8 weeks. Abbreviations are: Nx, nephrectomized; Eth, ethanol.The p-values in FIGS. 26A-26C represent statistically-significantdifferences from nephrectomized rats treated with PBS.

FIGS. 27A-27B show the output of an experiment evaluating the effect ofVB-703 on monocyte/macrophage cell infiltration in the glomeruli (FIG.27A) or in the interstitium (FIG. 27B). CD68 positive cells in theglomeruli (cells/glomeruli) and in the interstitium (cells/mm²) wereevaluated in healthy rats (n=3) (white bar), sham operated rats (n=3)(white bar with stripes), nephrectomized rats treated with solventcontrol (0.5% ethanol/PBS) (black bar) (n=7), nephrectomized rats VB-7034 mg/kg treated (n=7) (light gray bar), or nephrectomized ratstelmisartan 10 mg/kg treated (n=8) (dark gray bar) at 8 weeks.Abbreviations are: Nx, nephrectomized; Eth, ethanol.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining embodiments of the invention in detail, it is to beunderstood that the invention is not limited in its application to thedetails set forth in the following description or exemplified by theExamples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

General Definitions

The terms “comprises”, “comprising”, “includes”, “including”, “having”,and their conjugates mean “including but not limited to.”

The word “optionally” is used herein to mean “is provided in someembodiments and not provided in other embodiments.” Any particularembodiment of the invention can include a plurality of “optional”features unless such features conflict.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

As used herein, the term “about” modifying an amount related to theinvention refers to variation in the numerical quantity that can occur,for example, through routine testing and handling; through inadvertenterror in such testing and handling; through differences in themanufacture, source, or purity of ingredients employed in the invention;and the like. Whether or not modified by the term “about”, the claimsinclude equivalents of the recited quantities. In one embodiment, theterm “about” means within 10% of the reported numerical value.

The term “therapeutically effective amount,” as used herein, refers tothat amount of a given therapeutic agent sufficient to result inamelioration of one or more symptoms of a disorder or condition, orprevent appearance or advancement of a disorder or condition, or causeregression of or cure from the disorder or condition. In someembodiments, a therapeutically effective amount of the compounddescribed herein is about 5 mg to about 160 mg of the compound per day.

As used herein throughout, the term “alkyl” refers to a saturatedaliphatic hydrocarbon including straight chain and branched chaingroups. In some embodiments, the alkyl group has 1 to 20 carbon atoms.Whenever a numerical range; e.g., “1-20”, is stated herein, it impliesthat the group, in this case the alkyl group, may contain 1 carbon atom,2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbonatoms. In some embodiments, the alkyl is a medium size alkyl having 1 to10 carbon atoms. In some embodiments, the alkyl is a lower alkyl having1 to 4 carbon atoms. The alkyl group can be substituted (e.g., with 1 to5 substituent groups) or unsubstituted. In any of the embodimentsdescribed herein, the alkyl can be unsubstituted. In any of theembodiments described herein, the alkyl can also be substituted by oneto five substituent groups, wherein the substituent group can be, forexample, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide,sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, oxo, carbonyl,thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, andamino, as these terms are defined herein.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone of more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. Acycloalkyl group can be substituted (e.g., with 1 to 5 substituentgroups) or unsubstituted. In any of the embodiments described herein,the cycloalkyl can be unsubstituted. In any of the embodiments describedherein, the cycloalkyl can also be substituted by one to fivesubstituent groups, wherein the substituent group can be, for example,alkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo,hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy,sulfinyl, sulfonyl, cyano, nitro, azide, sulfonyl, sulfinyl,sulfonamide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea,thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as theseterms are defined herein.

An “alkenyl” group refers to an aliphatic hydrocarbon group whichcontains at least two carbon atoms and at least one carbon-carbon doublebond, which can be straight or branched. An alkenyl group can besubstituted or unsubstituted.

An “alkynyl” group refers to an aliphatic hydrocarbon group whichcontains at least two carbon atoms and at least one carbon-carbon triplebond. An alkynyl group can be substituted or unsubstituted.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. In any of theembodiments described herein, aryl groups can have 6 to 14 carbons,e.g., 6 to 10 carbons. Examples, without limitation, of aryl groups arephenyl, naphthalenyl and anthracenyl. The aryl group can be substituted(e.g., with 1 to 5 substituent groups) or unsubstituted. Whensubstituted, the substituent group can be, for example, alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy,alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl,sulfonyl, cyano, nitro, azide, sulfonyl, sulfinyl, sulfonamide,phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as these termsare defined herein. In any of the embodiments described herein, the arylgroup can be a phenyl group, optionally substituted, for example, by oneto five substituent such as halogens (e.g., fluorine or chlorine), alkylgroups (e.g., a C₁₋₄ alkyl), or halogen substituted alkyls (e.g.,trifluoromethyl).

A “heteroaryl” group refers to a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. In any ofthe embodiments described herein, heteroaryl groups can have 5 to 14ring atoms, e.g., 5 to 10 ring atoms (e.g., 5 or 6 ring atoms).Examples, without limitation, of heteroaryl groups include pyrrole,furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine,pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group canbe substituted (e.g., with 1 to 5 substituent groups) or unsubstituted.When substituted, the substituent group can be, for example, alkyl,alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo,hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy,sulfinyl, sulfonyl, cyano, nitro, azide, sulfonyl, sulfinyl,sulfonamide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea,thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as theseterms are defined herein.

A “heteroalicyclic” group refers to a monocyclic or fused ring grouphaving in the ring(s) one or more heteroatoms such as nitrogen, oxygenand sulfur. The rings may also have one or more double bonds. However,the rings do not have a completely conjugated pi-electron system. In anyof the embodiments described herein, heteroalicyclic groups can have 3to 10 ring atoms, e.g., 5 to 10 ring atoms (e.g., 5 or 6 ring atoms).The heteroalicyclic can be substituted (e.g., with 1 to 5 substituentgroups) or unsubstituted. When substituted, the substituted group canbe, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide,sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, oxo, carbonyl,thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, andamino, as these terms are defined herein. Representative examples arepiperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine andthe like.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group,wherein the alkyl or cycloalkyl can be any of those as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,wherein the aryl or heteroaryl can be any of those as defined herein.

A “thiohydroxy” group refers to a —SH group.

A “thioalkoxy” group refers to both an —S-alkyl group, and an—S-cycloalkyl group, wherein the alkyl or cycloalkyl can be any of thoseas defined herein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroarylgroup, wherein the aryl or heteroaryl can be any of those as definedherein.

A “carbonyl” group refers to a —C(═O)—R group, wherein R is hydrogen,alkyl, alkenyl, cycloalkyl, aryl, heteroaryl (bonded through a ringcarbon) or heteroalicyclic (bonded through a ring carbon) as definedherein.

An “aldehyde” group refers to a carbonyl group, wherein R is hydrogen.

A “thiocarbonyl” group refers to a —C(═S)—R group, wherein R is asdefined herein.

A “C-carboxy” group refers to a —C(═O)—O—R groups, wherein R is asdefined herein.

An “O-carboxy” group refers to an RC(═O)—O— group, wherein R is asdefined herein.

An “oxo” group refers to a ═O group.

A “carboxylic acid” group refers to a C-carboxyl group in which R ishydrogen.

A “halo” group or “halogen” refers to fluorine, chlorine, bromine oriodine.

A “trihalomethyl” group refers to a —CX₃ group wherein X is a halo groupas defined herein, e.g., a CF₃ group.

A “sulfinyl” group refers to an —S(═O)—R group, wherein R is as definedherein.

A “sulfonyl” group refers to an —S(═O)₂—R group, wherein R is as definedherein.

An “S-sulfonamido” group refers to a —S(═O)₂—NR₂ group, with each of Ras is defined herein.

An “N-sulfonamido” group refers to an RS(═O)₂—NR group, wherein each ofR is as defined herein.

An “O-carbamyl” group refers to an —OC(═O)—NR₂ group, wherein each of Ris as defined herein.

An “N-carbamyl” group refers to an ROC(═O)—NR— group, wherein each of Ris as defined herein.

An “O-thiocarbamyl” group refers to an —OC(═S)—NR₂ group, wherein eachof R is as defined herein.

An “N-thiocarbamyl” group refers to an ROC(═S)NR— group, wherein each ofR is as defined herein.

An “amino” group refers to an —NR₂ group wherein each of R is as definedherein.

A “C-amido” group refers to a —C(═O)—NR₂ group, wherein each of R is asdefined herein.

An “N-amido” group refers to an RC(═O)—NR— group, wherein each of R isas defined herein.

An “urea” group refers to an —NRC(═O)—NR₂ group, wherein each of R is asdefined herein.

A “guanidino” group refers to an —RNC(═N)—NR₂ group, wherein each of Ris as defined herein.

A “guanyl” group refers to an R₂NC(═N)— group, wherein each of R is asdefined herein.

The term “phosphonyl” or “phosphonate” describes a —P(═O)(OR)₂ group,with R as defined herein.

The term “phosphate” describes an —O—P(═O)(OR)₂ group, with each of R asdefined herein.

A “phosphoric acid” is a phosphate group wherein each of R is hydrogen.

The term “phosphinyl” describes a —PR₂ group, with each of R as definedherein.

The term “thiourea” describes a —NR—C(═S)—NR— group, with each of R asdefined herein.

The term “saccharide” refers to one or more sugar units, either anopen-chain sugar unit or a cyclic sugar unit (e.g., pyranose- orfuranose-based units), and encompasses any monosaccharide, disaccharideand oligosaccharide, unless otherwise indicated.

The term “stereoisomer” includes geometric isomers, such as E or Zisomers, enantiomers, diastereomers, and the like.

The term “stereoisomeric mixture” includes any mixture in any ratio ofstereoisomers defined herein. In some embodiments, a stereoisomericmixture includes a racemic mixture. In some embodiments, astereoisomeric mixture includes an enantiomerically enriched mixture. Insome embodiments, a stereoisomeric mixture includes a mixture ofdiastereomers in any ratio.

The term “enantiomeric excess” or “ee” refers to a measure for how muchof one enantiomer is present compared to the other. For a mixture of Rand S enantiomers, the percent enantiomeric excess is defined as|R−S|*100, where R and S are the respective mole or weight fractions ofenantiomers in a mixture such that R+S=1. With knowledge of the opticalrotation of a chiral substance, the percent enantiomeric excess isdefined as ([α]_(obs)/[α]_(max))*100, where [α]_(obs) is the opticalrotation of the mixture of enantiomers and [α]_(max) is the opticalrotation of the pure enantiomer.

The term “salt” includes both internal salt or external salt. In someembodiments, the salt is an internal salt, i.e., a zwitterion structure.In some embodiments, the salt is an external salt. In some embodiments,the external salt is a pharmaceutically acceptable salt having asuitable counter ion. Suitable counterions for pharmaceutical use areknown in the art.

Throughout this application, various embodiments of this invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range, such as from 1 to 6 should be considered to havespecifically disclosed subranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 3, 4, 5, and 6.This applies regardless of the breadth of the range.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Oxidized Lipids

The present invention is directed, in part, to oxidized lipid compounds.

In some embodiments, an oxidized lipid of the invention is a compoundaccording to Formula 1:

wherein each of B₁, B₂, and B₃ is independently selected from the groupconsisting of oxygen, sulfur, nitrogen, phosphorus and silicon, whereineach of said nitrogen, phosphorus and silicon is optionally substitutedby one or more substituents selected from the group consisting of alkyl,halo, cycloalkyl, aryl, hydroxy, thiohydroxy, alkoxy, aryloxy,thioaryloxy, thioalkoxy and oxo;

wherein R¹⁰ is a C₂₋₂₈ alkyl optionally substituted by one to five R¹¹substituents, wherein each R¹¹ is independently selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, phosphonate, phosphate, phosphinyl, sulfonyl, sulfinyl,sulfonamide, amide, carbonyl, thiocarbonyl, C-carboxy, O-carboxy,C-carbamate, N-carbamate, C-thiocarboxy, S-thiocarboxy, and amino;

wherein p is an integer selected from 1-10;

wherein q is an integer selected from 1-26;

wherein R²⁰ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; and

wherein Het is a heteroalicyclic ring or a heteroaryl. In otherembodiments, the oxidized lipid is a compound according to Formula 1, ora stereoisomer, a stereoisomeric mixture, or a salt thereof. In someembodiments, the oxidized lipid having a structure according to Formula1 has a zwittionic structure. In some embodiments, the oxidized lipid isan external salt (e.g., a pharmaceutically acceptable salt) of thecompound having a structure according to Formula 1. In some embodiments,the compound according to Formula 1 has a structure according to Formula1a or Formula 1b:

or a salt thereof.

In some embodiments, the compound has a structure according to Formula1a, wherein the compound has an enantiomeric purity of about 80% ee ormore, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee,about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee,about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more. Insome embodiments, the compound has a structure according to Formula 1b,wherein the compound has an enantiomeric purity of about 80% ee or more,e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92%ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97%ee, about 98% ee, about 99% ee, about 99.5% ee or more. In otherembodiments, the compound has an enantiomeric purity of from about 80%ee to about 100% ee, about 85% ee to about 100% ee, about 90% ee toabout 100% ee, about 95% ee to about 100%, about 80% ee to about 99.5%ee, about 85% ee to about 99.5% ee, about 90% ee to about 99.5% ee,about 95% ee to about 99.5%, or any range thereof.

Suitable B₁, B₂, and B₃ for Formula 1 are defined herein. In someembodiments, B₁ is O. In some embodiments, B₂ is O. In some embodiments,B₃ is O. In some embodiments, at least two of B₁, B₂, and B₃ are O,e.g., B₁, B₂ are O and B₃ is O or S; B₁, B₃ are O and B₂ is O or S; orB₂, B₃ are O and B₁ is O or S. In some embodiments, B₁ is S. In someembodiments, B₂ is S. In some embodiments, B₃ is S. In some embodiments,at least two of B₁, B₂, and B₃ are S, e.g., B₁, B₂ are S and B₃ is O orS; B₁, B₃ are S and B₂ is O or S; or B₂, B₃ are S and B₁ is O or S. Insome embodiments, all of B₁, B₂, and B₃ are O. In some embodiments, allof B₁, B₂, and B₃ are S.

Suitable R¹⁰ for Formula 1 are defined herein. In some embodiments, R¹⁰is a C₂₋₂₈ alkyl. In some embodiments, R¹⁰ is a straight chain C₂₋₂₈alkyl, e.g., an alkyl chain having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbons,substituted or unsubstituted. In some embodiments, R¹⁰ is a straightchain C₂₋₂₈ alkyl, e.g., an alkyl chain having 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28carbons, substituted by one to five R¹¹ substituents, wherein each R¹¹is independently as defined herein, e.g., a halogen (e.g., F) or analkyl (e.g., a C₁₋₁₀ alkyl). In some embodiments, R¹⁰ is selected fromthe group consisting of hexadecyl, dodecyl, octadecyl, octyl, eicosanyl,cis-9-hexadecenyl, (2′-octyl)dodecyl, and (15′-carboxy)pentadecyl. Insome embodiments, R¹⁰ is hexadecyl. In some embodiments, R¹⁰ is(2′-octyl)dodecyl. In some embodiments, R¹⁰ is eicosanyl.

Suitable R²⁰ for Formula 1 are defined herein. In some embodiments, R²⁰is a hydrogen or an alkyl. In some embodiments, R²⁰ is hydrogen. In someembodiments, R²⁰ is an alkyl, e.g., a C₁₋₄ alkyl (e.g., methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, or tert-butyl). Insome embodiments, R²⁰ is methyl.

Suitable values for p and q in Formula 1 are defined herein. In someembodiments, q is an integer of 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10. In some embodiments, q is 4. In some embodiments, p is an integerof 1-7, e.g., 1, 2, 3, 4, 5, 6, or 7. In some embodiments, p is 2.

Suitable Het for Formula 1 are defined herein. In some embodiments, Hetis a heteroaryl. In some embodiments, Het is a monocyclic heteroaryl. Insome embodiments, Het is a nitrogen containing heteroaryl (e.g.,monocyclic heteroaryl). In some embodiments, Het is a monocyclicheteroaryl containing 1, 2, 3, or 4 nitrogen atoms. In some embodiments,Het is a 6-member ring monocyclic heteroaryl, e.g., pyridine,pyrimidine, pyridazine, pyrazine, triazine, etc. In some embodiments,Het is a 5-member ring monocyclic heteroaryl, e.g., imidazole, thiazole,isothiazole, oxazole, isoxazole, oxidiazole, pyrazole, triazole, etc. Insome embodiments, Het is a bicyclic heteroaryl containing nitrogenatoms, e.g., 1-3 nitrogen atoms, e.g., quinoline, isoquinoline,quinazoline, thienopyridine, thienopyrimidine, pyrrolopyridine,imidazopyridine, etc. In any of the embodiments described herein, Hetcan be a nitrogen containing heteroaryl, wherein a nitrogen atom of theheteroaryl is directly connected to the alkylene chain, i.e.,—(CH₂)_(p)— in Formula 1 to form a cation. In some embodiments, Het ispyridine, wherein the nitrogen atom of the pyridine is directlyconnected to the alkylene chain, i.e., —(CH₂)_(p)— in Formula 1 form apyridinium salt (e.g., an internal salt or an external salt as describedherein). In some embodiments, Het is an unsubstituted pyridine, whereinthe nitrogen atom of the pyridine is directly connected to the alkylenechain, i.e., —(CH₂)_(p)— in Formula 1. In some embodiments, Het is asubstituted pyridine, wherein the nitrogen atom of the pyridine isdirectly connected to the alkylene chain, i.e., —(CH₂)_(p)— in Formula1, wherein the pyridine is substituted by one to five (e.g., 1, 2, 3, 4,or 5) R¹² substituents, wherein each R¹² is independently selected fromthe group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroaryl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, phosphonate, phosphate, phosphinyl, sulfonyl,sulfinyl, sulfonamide, amide, carbonyl, thiocarbonyl, C-carboxy,O-carboxy, C-carbamate, N-carbamate, C-thiocarboxy, S-thiocarboxy, andamino as defined herein. In some embodiments, each R¹² is independently,e.g., a halogen (e.g., F, Cl), a C₆₋₁₀ aryl (e.g., phenyl), aheteroaryl, or an alkyl (e.g., a C₁₋₁₀ alkyl, e.g., a C₁₋₄ alkyl (e.g.,methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl,tert-butyl). In some embodiments, Het is a substituted pyridine and thepyridine is substituted by one R¹² substituent at the 2-, 3-, or4-position of the pyridine, wherein R¹² is as defined herein, forexample, a halogen (e.g., F, Cl), a phenyl, or a methyl.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 2:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof. SuitableR¹⁰, R²⁰, p, q, and Het are those as defined herein for Formula 1. Insome embodiments, the oxidized lipid having a structure according toFormula 2 has a zwittionic structure. In some embodiments, the oxidizedlipid is an external salt (e.g., a pharmaceutically acceptable salt) ofthe compound having a structure according to Formula 2. In someembodiments, the compound according to Formula 2 has a structureaccording to Formula 2a or Formula 2b:

or a salt thereof.

In some embodiments, the compound is a S-isomer having a structureaccording to Formula 2a, wherein the compound has an enantiomeric purityof about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee,about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee,about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% eeor more. In some embodiments, the compound is a R-isomer having astructure according to Formula 2b, wherein the compound has anenantiomeric purity of about 80% ee or more, e.g., about 80% ee, about85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about99% ee, about 99.5% ee or more. In other embodiments, the compound hasan enantiomeric purity of from about 80% ee to about 100% ee, about 85%ee to about 100% ee, about 90% ee to about 100% ee, about 95% ee toabout 100%, about 80% ee to about 99.5% ee, about 85% ee to about 99.5%ee, about 90% ee to about 99.5% ee, about 95% ee to about 99.5%, or anyrange thereof.

In some embodiments according to Formula 2, R¹⁰ is selected from thegroup consisting of hexadecyl, dodecyl, octadecyl, octyl, eicosanyl,cis-9-hexadecenyl, (2′-octyl)dodecyl, and (15′-carboxy)pentadecyl; R²⁰is hydrogen or an alkyl, e.g., a C₁₋₄ alkyl (e.g., methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, or tert-butyl); q isan integer of 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is aninteger of 1-7, e.g., 1, 2, 3, 4, 5, 6, or 7; and Het is anunsubstituted pyridine, wherein the nitrogen atom of the pyridine isdirectly connected to the alkylene chain, i.e., —(CH₂)_(p)— in Formula2, or Het is a substituted pyridine, wherein the nitrogen atom of thepyridine is directly connected to the alkylene chain, i.e., —(CH₂)_(p)—in Formula 2, wherein the pyridine is substituted by one to five (e.g.,1, 2, 3, 4, or 5) R¹² substituents, wherein each R¹² is independently asdefined herein, e.g., a halogen (e.g., F, Cl), a C₆₋₁₀ aryl (e.g.,phenyl), a heteroaryl, or an alkyl (e.g., a C₁₋₁₀ alkyl, e.g., a C₁₋₄alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, tert-butyl). In some embodiments, Het is a substitutedpyridine and the pyridine is substituted by one R¹² substituent at the2-, 3-, or 4-position of the pyridine, wherein R¹² is as defined herein,for example, a halogen (e.g., F, Cl), a phenyl, or a methyl.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 3:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof, whereinR¹⁰, R¹², R²⁰, p, and q are as defined herein for Formula 1. In someembodiments, the oxidized lipid is a zwittionic structure according toFormula 3. In some embodiments, the oxidized lipid is an external salt(e.g., a pharmaceutically acceptable salt) of the compound having astructure according to Formula 3. In some embodiments, the oxidizedlipid according to Formula 3 has a structure according to Formula 3a orFormula 3b:

or a salt thereof.

In some embodiments, the compound is a S-isomer having a structureaccording to Formula 3a, wherein the compound has an enantiomeric purityof about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee,about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee,about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% eeor more. In some embodiments, the compound is a R-isomer having astructure according to Formula 3b, wherein the compound has anenantiomeric purity of about 80% ee or more, e.g., about 80% ee, about85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about99% ee, about 99.5% ee or more. In other embodiments, the compound hasan enantiomeric purity of from about 80% ee to about 100% ee, about 85%ee to about 100% ee, about 90% ee to about 100% ee, about 95% ee toabout 100%, about 80% ee to about 99.5% ee, about 85% ee to about 99.5%ee, about 90% ee to about 99.5% ee, about 95% ee to about 99.5%, or anyrange thereof.

In some embodiments according to Formula 3, R¹⁰ is selected from thegroup consisting of hexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ ishydrogen or a C₁₋₄ alkyl (e.g., methyl, ethyl, propyl, isopropyl,n-butyl, sec-butyl, iso-butyl, or tert-butyl); q is an integer of 2-6,e.g., 2, 3, 4, 5, or 6; p is an integer of 2-5, e.g., 2, 3, 4, or 5; andthe pyridine is substituted by 0 to 3 (e.g., 0, 1, 2, or 3) R¹²substituents, wherein each R¹² is independently as defined herein, e.g.,a halogen (e.g., F, Cl), a C₆₋₁₀ aryl (e.g., phenyl), a heteroaryl, or aC₁₋₄ alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, tert-butyl). In some embodiments, R¹⁰ is selected from thegroup consisting of hexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ ishydrogen or methyl; q is 2, 3, 4, 5, or 6; p is 2, 3, 4, or 5; and thepyridine is substituted by 0 to 3 (e.g., 0, 1, 2, or 3) R¹²substituents, wherein each R¹² is independently is a halogen (e.g., F,Cl), a C₆₋₁₀ aryl (e.g., phenyl), a heteroaryl, or a C₁₋₄ alkyl (e.g.,methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl,tert-butyl). In some embodiments, R¹⁰ is selected from the groupconsisting of hexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ ishydrogen or methyl; q is 4; p is 2; and the pyridine is substituted by0, 1, or 2 R¹² substituents, wherein each R¹² is independently a halogen(e.g., F, Cl), a phenyl, or a methyl. In some embodiments according toFormulae 3, 3a, and 3b, R¹⁰ is selected from the group consisting ofhexadecyl, eicosanyl, and (2′-octyl)dodecyl. In some embodimentsaccording to Formulae 3, 3a, and 3b, R²⁰ is a hydrogen or a C₁₋₄ alkyl.In some embodiments according to Formulae 3, 3a, and 3b, q is an integerof 2-6. In some embodiments according to Formulae 3, 3a, and 3b, p is aninteger of 2-5. In some embodiments according to Formulae 3, 3a, and 3b,the pyridine is substituted by 0 to 3 (e.g., 0, 1, 2, or 3) R¹²substituents, wherein each R¹² is independently as defined herein, e.g.,a halogen (e.g., F, Cl), a C₆₋₁₀ aryl (e.g., phenyl), a heteroaryl, or aC₁₋₄ alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, tert-butyl). In some embodiments according to Formulae 3, 3a,and 3b, the pyridine ring is substituted by one, two, or three R¹²substituents. In some embodiments according to Formulae 3, 3a, and 3b,the pyridine ring is substituted by one R¹² substituent, wherein the oneR¹² substituent is a fluorine or a phenyl, e.g., the pyridine ring is3-fluoro-pyridine or 3-phenyl-pyridine. In some embodiments according toFormulae 3, 3a, and 3b, R¹⁰ is selected from the group consisting ofhexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ is hydrogen or methyl; qis 4; p is 2; and the pyridine is 3-fluoro-pyridine or3-phenyl-pyridine.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 4:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof. SuitableR¹⁰, R²⁰, and q are those as defined herein for Formula 1. In someembodiments, the oxidized lipid has a zwittionic structure according toFormula 4. In some embodiments, the oxidized lipid is an external salt(e.g., a pharmaceutically acceptable salt) of the compound having astructure according to Formula 4. In some embodiments, the oxidizedlipid according to Formula 4 has a structure according to Formula 4a orFormula 4b:

or a salt thereof.

In some embodiments, the compound is a S-isomer having a structureaccording to Formula 4a, wherein the compound has an enantiomeric purityof about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee,about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee,about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% eeor more. In some embodiments, the compound is a R-isomer having astructure according to Formula 4b, wherein the compound has anenantiomeric purity of about 80% ee or more, e.g., about 80% ee, about85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about99% ee, about 99.5% ee or more. In other embodiments, the compound hasan enantiomeric purity of from about 80% ee to about 100% ee, about 85%ee to about 100% ee, about 90% ee to about 100% ee, about 95% ee toabout 100%, about 80% ee to about 99.5% ee, about 85% ee to about 99.5%ee, about 90% ee to about 99.5% ee, about 95% ee to about 99.5%, or anyrange thereof.

In some embodiments according to Formula 4, R¹⁰ is selected from thegroup consisting of hexadecyl, dodecyl, octadecyl, octyl, eicosanyl,cis-9-hexadecenyl, (2′-octyl)dodecyl, and (15′-carboxy)pentadecyl. Insome embodiments, R¹⁰ is hexadecyl. In some embodiments, R¹⁰ is(2′-octyl)dodecyl. In some embodiments, R¹⁰ is eicosanyl.

In some embodiments, R²⁰ is a hydrogen or an alkyl. In some embodiments,R²⁰ is hydrogen. In some embodiments, R²⁰ is an alkyl, e.g., a C₁₋₄alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, or tert-butyl). In some embodiments, R²⁰ is methyl.

In some embodiments, q is an integer of 1-10, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10. In some embodiments, q is 4.

In some embodiments, R¹⁰ is selected from the group consisting ofhexadecyl, eicosanyl, and (2′-octyl)dodecyl; R²⁰ is hydrogen or a C₁₋₄alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, or tert-butyl); and q is an integer of 2-6, e.g., 2, 3, 4, 5,or 6. In some embodiments, R¹⁰ is selected from the group consisting ofhexadecyl, eicosanyl and (2′-octyl)dodecyl; R²⁰ is hydrogen; and q is aninteger of 2-6, e.g., 2, 3, 4, 5, or 6. In some embodiments, R¹⁰ isselected from the group consisting of hexadecyl, eicosanyl, and(2′-octyl)dodecyl; R²⁰ is methyl; and q is an integer of 2-6, e.g., 2,3, 4, 5, or 6.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 5,

or a stereoisomer, a stereoisomeric mixture, or a salt thereof, whereinR¹⁰ and R²⁰ are as defined herein for Formula 1. In some embodiments,the oxidized lipid has a zwittionic structure according to Formula 5. Insome embodiments, the oxidized lipid is an external salt (e.g., apharmaceutically acceptable salt) of the compound having a structureaccording to Formula 5. In some embodiments, the oxidized lipid is acompound according to Formula 5 has a structure according to Formula 5aor Formula 5b:

or a salt thereof.

In some embodiments, the compound is a S-isomer having a structureaccording to Formula 5a, wherein the compound has an enantiomeric purityof about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee,about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee,about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% eeor more. In some embodiments, the compound is a R-isomer having astructure according to Formula 5b, wherein the compound has anenantiomeric purity of about 80% ee or more, e.g., about 80% ee, about85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about99% ee, about 99.5% ee or more. In other embodiments, the compound hasan enantiomeric purity of from about 80% ee to about 100% ee, about 85%ee to about 100% ee, about 90% ee to about 100% ee, about 95% ee toabout 100%, about 80% ee to about 99.5% ee, about 85% ee to about 99.5%ee, about 90% ee to about 99.5% ee, about 95% ee to about 99.5%, or anyrange thereof.

In some embodiments according to Formula 5, R¹⁰ is selected from thegroup consisting of hexadecyl, dodecyl, octadecyl, octyl, eicosanyl,cis-9-hexadecenyl, (2′-octyl)dodecyl, and (15′-carboxy)pentadecyl. Insome embodiments, R¹⁰ is hexadecyl. In some embodiments, R¹⁰ is(2′-octyl)dodecyl. In some embodiments, R¹⁰ is eicosanyl.

In some embodiments, R²⁰ is a hydrogen or an alkyl. In some embodiments,R²⁰ is hydrogen. In some embodiments, R²⁰ is an alkyl, e.g., a C₁₋₄alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,iso-butyl, or tert-butyl). In some embodiments, R²⁰ is methyl.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure of:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof.

In some embodiments, the invention provides a compound selected from thegroup consisting of(R)-1-hexadecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-701);(R)-1-eicosanyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-702);(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-703);(R)-1-hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-704); and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoricacid pyridiniumethyl ester (VB-705). The prefix “(R)-” refers to theconfiguration of the C-2 carbon of the glycerol backbone. In someembodiments, the compound has an enantiomeric purity of about 80% ee ormore, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee,about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee,about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure of:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof.

In some embodiments, the invention provides a compound selected from thegroup consisting of(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-fluoro-pyridiniumethyl ester (VB-706) and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-phenyl-pyridiniumethyl ester (VB-707). The prefix “(R)-” refers to theconfiguration of the C-2 carbon of the glycerol backbone. In someembodiments, the compound has an enantiomeric purity of about 80% ee ormore, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee,about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee,about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more. Inother embodiments, the compound has an enantiomeric purity of from about80% ee to about 100% ee, about 85% ee to about 100% ee, about 90% ee toabout 100% ee, about 95% ee to about 100%, about 80% ee to about 99.5%ee, about 85% ee to about 99.5% ee, about 90% ee to about 99.5% ee,about 95% ee to about 99.5%, or any range thereof.

In other embodiments, an oxidized lipid compound of the invention treatsor prevents fibrosis (e.g., liver fibrosis, kidney fibrosis, focal andsegmental glomerulosclerosis, or any other fibrosis described herein) aswell as, or better than, telmisartan. In other embodiments, an oxidizedlipid compound of the invention reduces liver inflammation as well as,or better than, telmisartan. In other embodiments, an oxidized lipidcompound of the invention reduces liver fibrosis as well as, or betterthan, telmisartan. In other embodiments, an oxidized lipid compound ofthe invention treats or prevents kidney fibrosis as well as, or betterthan, telmisartan. In other embodiments, an oxidized lipid compound ofthe invention treats or prevents focal and segmental glomerulosclerosisas well as, or better than, telmisartan.

In other embodiments, an oxidized lipid compound of the inventioninhibits formation of ligand-induced phosphorylation of IKK, ERK, AKT,or p38 comparable to or more than VB-201(1-hexadecyl-2-(4′-carboxy)butyl-glycero-3-phosphocholine or1-hexadecyl-2-(4′-carboxybutyl)-glycerol-3-phosphocholine)) inhibitsformation of ligand-induced phosphorylation of IKK, ERK, AKT, or p38. Inother embodiments, an oxidized lipid compound of the invention inhibitsligand-induced cell migration comparable to or more than VB-201 inhibitsligand-induced cell migration. In other embodiments, the ligand is LPS,PGN, PAM3, or MCP1. In other embodiments, the cell migration is monocytemigration.

Methods of Synthesis

Other embodiments of the invention relate to methods of synthesizingoxidized lipids of the invention.

In some embodiments, the invention provides a method of synthesizing acompound of Formula 1,

or a stereoisomer, a stereoisomeric mixture, or a salt thereof, whereinB₁, B₂, B₃, R¹⁰, R²⁰ , Het, p, and q are defined herein above forFormula 1,the method comprising

a) reacting Intermediate 1

with Het to form the compound of Formula 1,

wherein B₁, B₂, B₃, R¹⁰, Het, p, and q are as defined above in Formula1; and wherein LG is a leaving group. In some embodiments, R^(20′) isthe same as R²⁰. However, as described in the Examples section, whenIntermediate 1 was heated in pyridine (e.g., at reflux temperature),conversion of —COOR^(20′) into —COOH was also observed. Thus, in someembodiments, R²⁰ is H, R^(20′) is selected from the group consisting ofalkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl, the reactingof Intermediate 1 with Het (e.g., pyridine or substituted pyridine asdefined herein) also converts —COOR^(20′) into —COOH, or a salt thereof,to form the compound of Formula 1. In other embodiments, R²⁰ is H,R^(20′) is selected from the group consisting of alkyl, alkenyl,alkynyl, cycloalkyl, aryl, and heteroaryl, the reacting of Intermediate1 with Het is followed by a step of hydrolysis to convert —COOR^(20′)into—COOH, or a salt thereof, to form the compound of Formula 1.Inpreferred embodiments, R²⁰ is H, R^(20′) is methyl, the reacting ofIntermediate 1 with Het (e.g., pyridine or substituted pyridine asdefined herein) also converts —COOR^(20′) into —COOH, or a salt thereof,to form the compound of Formula 1. In some embodiments, the LG isselected from the group consisting of halogen and oxygen containingleaving groups (e.g., as described herein).

In some embodiments, the Intermediate 1 can be synthesized by

b) reacting Reactant 1

with Reactant 2

to form a monochloro reaction product; andc) hydrolyzing the monochloro reaction product to form Intermediate 1,wherein B₁, B₂, B₃, R¹⁰, R20′, p, q, and LG are as defined above inIntermediate 1.

In other embodiments, the Intermediate 1 can be synthesized by

b′) reacting Reactant 1

with POCl₃ to form a POCl₃ reaction product;

-   c′) reacting Reactant 2A

-    with the POCl₃ reaction product to form a second reaction product;    and-   d′) hydrolyzing the second reaction product to form Intermediate 1,-   wherein B₁, B₂, B₃, R¹⁰, R^(20′), p, q, and LG are as defined above    in Intermediate 1. In some embodiments, steps b′) and c′) can be    performed in a one-pot fashion. In some embodiments, the POCl₃    reaction product is not isolated before reacting with Reactant 2A.

Suitable LGs for the methods of synthesis described herein include anyleaving group known in the art. In some embodiments, LG is a halogen,such as F, Cl, Br, or I. In some embodiments, LG is an oxygen containingleaving group, such as a tosylate, a mesylate, a triflate, etc. In someembodiments, LG is Br. Oxygen containing leaving groups as used hereinrefer to leaving groups represented by formula

wherein G is typically an electron withdrawing group, e.g.,

Examples of oxygen containing leaving groups include sulfonates such asnonaflate, triflate, fluorosulfonate, tosylate, mesylate or besylate.Other oxygen containing leaving groups such as acyloxy and aryloxygroups are known in the art.

In some embodiments, the LG is a leaving group selected from the groupconsisting of oxygen containing leaving groups represented by

wherein R¹⁰⁰ is selected from the group consisting of alkyl, alkenyl,alkynyl, cycloalkyl, heteroalicycloalkyl, aryl, and heteroaryl, each ofwhich is optionally substituted by one to five R¹³, wherein each R¹³substituent is independently selected from the group consisting ofalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, halo,trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, phosphonate, phosphate, phosphinyl, sulfonyl, sulfinyl,sulfonamide, amide, carbonyl, thiocarbonyl, C-carboxy, O-carboxy,C-carbamate, N-carbamate, C-thiocarboxy, S-thiocarboxy, and amino. Insome embodiments, R¹⁰⁰ is p-tolyl, o-tolyl, phenyl, methyl, ortrifluoromethyl. In some embodiments, R¹⁰⁰ is p-tolyl.

In some embodiments, all of B₁, B₂, and B₃ are O. In some embodiments,Het is an unsubstituted pyridine, wherein the nitrogen atom of thepyridine is directly connected to the alkylene chain, i.e., —(CH₂)_(p)—in Formula 1. In some embodiments, Het is 3-fluoro-pyridine or3-phenyl-pyridine, wherein the nitrogen atom of the pyridine is directlyconnected to the alkylene chain, i.e., —(CH₂)_(p)— in Formula 1. In someembodiments, R¹⁰ is selected from the group consisting of hexadecyl,eicosanyl and (2′-octyl)dodecyl. In some embodiments, R²⁰ is hydrogen ora C₁₋₄ alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, iso-butyl, or tert-butyl). In some embodiments, p is 2. Insome embodiments, q is 4.

In some embodiments, the method described above is directed tosynthesizing compounds having a structure according to Formulae 2, 3, 4,or 5 (e.g., as described herein). In such embodiments, all of B₁, B₂,and B₃ are O, and suitable R¹⁰, R¹¹, R¹², R²⁰, Het, p, and q for themethods described above are those as defined herein for the respectiveFormulae 2, 3, 4, or 5. In some embodiments, R^(20′) is the same as R²⁰.In some embodiments, R²⁰ is H, R^(20′) is selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl,the reacting of Intermediate 1 with Het (e.g., pyridine or substitutedpyridine as defined herein) also converts —COOR^(20′) into —COOH, or asalt thereof, to form the compound of Formulae 2, 3, 4, or 5. In otherembodiments, R²⁰ is H, R^(20′) is selected from the group consisting ofalkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl, the reactingof Intermediate 1 with Het is followed by a step of hydrolysis toconvert —COOR^(20′) into —COOH, or a salt thereof, to form the compoundof Formulae 2, 3, 4, or 5. In preferred embodiments, R²⁰ is H inFormulae 2, 3, 4, or 5, R^(20′) is methyl, the reacting of Intermediate1 with Het (e.g., pyridine or substituted pyridine as defined herein)also converts —COOR^(20′) into —COOH, or a salt thereof, to form thecompound of Formulae 2, 3, 4, or 5.

Suitable solvents, reaction temperatures, as well as other reactionparameters for each of the process steps described herein can beascertained by those skilled in the art based on the working Examplesdescribed herein.

Thus, in some embodiments, a compound having a structure according toFormula 1 (e.g., as described herein) can be synthesized by a methodcomprising the steps in Method A:

Suitable LGs include any leaving group known in the art. In someembodiments, LG is a halogen, such as F, Cl, Br, or I. In someembodiments, LG is an oxygen containing leaving group, such as atosylate, a mesylate, a triflate, etc. In some embodiments, LG is Br. Insome embodiments, LG is tosylate.

In some embodiments, a compound having a structure according to Formula1 can also be synthesized by a method comprising the steps in Method B:

Method B

Suitable R¹⁰⁰ for Method B include alkyl, alkenyl, alkynyl, cycloalkyl,heteroalicycloalkyl, aryl, or heteroaryl, optionally substituted by oneto five R¹³, wherein each R¹³ substituent is independently selected fromthe group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroaryl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, phosphonate, phosphate, phosphinyl, sulfonyl,sulfinyl, sulfonamide, amide, carbonyl, thiocarbonyl, C-carboxy,O-carboxy, C-carbamate, N-carbamate, C-thiocarboxy, S-thiocarboxy, andamino as described herein. In some embodiments, R¹⁰⁰ is a p-tolyl,o-tolyl, phenyl, methyl, or trifluoromethyl moiety. In some embodiments,R¹⁰⁰ is p-tolyl.

In some embodiments, the method for synthesizing a compound having astructure according to Formula 1 further comprises a step of hydrolysisto convert an ester into a carboxylic acid.

Suitable B₁, B₂, B₃, R¹⁰, R¹¹, R¹², R²⁰, Het, p, and q for Method A or Bare those as defined herein for Formula 1. In some embodiments, all ofB₁, B₂, and B₃ are O. In some embodiments, Het is an unsubstitutedpyridine, wherein the nitrogen atom of the pyridine is directlyconnected to the alkylene chain, i.e., —(CH₂)_(p)— in Formula 1. In someembodiments, R¹⁰ is selected from the group consisting of hexadecyl,eicosanyl and (2′-octyl)dodecyl. In some embodiments, R²⁰ is hydrogen ora C₁₋₄ alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, iso-butyl, or tert-butyl). In some embodiments, p is 2. Insome embodiments, q is 4. In some embodiments, R^(20′) is the same asR²⁰. In some embodiments, R²⁰ is H, R^(20′) is selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl,the reacting of Intermediate 1 with Het (e.g., pyridine or substitutedpyridine as defined herein) also converts —COOR^(20′) into —COOH, or asalt thereof, to form the compound of Formula 1. In other embodiments,R²⁰ is H, R^(20′) is selected from the group consisting of alkyl,alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl, the reacting ofIntermediate 1 with Het is followed by a step of hydrolysis to convert—COOR^(20′) into —COOH, or a salt thereof, to form the compound ofFormula 1. In preferred embodiments, R²⁰ is H, R^(20′) is methyl, thereacting of Intermediate 1 with Het (e.g., pyridine or substitutedpyridine as defined herein) also converts —COOR^(20′) into —COOH, or asalt thereof, to form the compound of Formula 1.

Similarly, compounds having a structure according to Formulae 2, 3, 4,or 5 (e.g., as described herein) can be synthesized by any of themethods described above, e.g., by a method comprising the steps inMethod A or the steps in Method B. In the synthesis of compounds havinga structure according to Formulae 2, 3, 4, or 5 (e.g., as describedherein), all of B₁, B₂, and B₃ are O, and suitable R¹⁰, R¹¹, R¹², R²⁰, ,Het, p, and q for Method A or B are those as defined herein for therespective Formulae 2, 3, 4, or 5. In some embodiments, R^(20′) is thesame as R²⁰. In some embodiments, R²⁰ is H, R^(20′) is selected from thegroup consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, andheteroaryl, the reacting of Intermediate 1 with Het (e.g., pyridine orsubstituted pyridine as defined herein) also converts —COOR^(20′) into—COOH, or a salt thereof, to form the compound of Formulae 2, 3, 4, or5. In other embodiments, R²⁰ is H, R^(20′) is selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl,the reacting of Intermediate 1 with Het is followed by a step ofhydrolysis to convert —COOR^(20′) into —COOH, or a salt thereof, to formthe compound of Formulae 2, 3, 4, or 5. In preferred embodiments, R²⁰ isH, R^(20′) is methyl, the reacting of Intermediate 1 with Het (e.g.,pyridine or substituted pyridine as defined herein) also converts—COOR^(20′) into —COOH, or a salt thereof, to form the compound ofFormulae 2, 3, 4, or 5.

In some embodiments, the invention provides a method of synthesizing acompound of Formula 1,

or a stereoisomer, a stereoisomeric mixture, or a salt thereof,the method comprising

a) reacting Reactant 10

with R²⁰OH to form the compound of Formula 1,

wherein B₁, B₂, B₃, R¹⁰, Het, p, and q are defined above; and whereinR²⁰ is selected from the group consisting of alkyl, alkenyl, alkynyl,cycloalkyl, aryl, and heteroaryl. Preferably, R²⁰ is an alkyl (e.g.,C₁₋₄ alkyl, e.g., methyl). In some embodiments, the reaction iscatalyzed by an acid, e.g., HCl. Other known methods for forming anester can also be used in step a).

In some embodiments, the method is directed to synthesizing compoundshaving a structure according to Formulae 2, 3, 4, or 5 (e.g., asdescribed herein). In such embodiments, all of B₁, B₂, and B₃ are O, andsuitable R¹⁰, R¹¹, R¹², Het, p, and q for the methods described aboveare those as defined herein for the respective Formulae 2, 3, 4, or 5,wherein R²⁰ is selected from the group consisting of alkyl, alkenyl,alkynyl, cycloalkyl, aryl, and heteroaryl. Preferably, R²⁰ is an alkyl(e.g., C₁₋₄ alkyl, e.g., methyl).

Oxidized lipids of the invention (e.g., Formulae 1a, 1b, 2a, 2b, 3a, 3b,4a, 4b, 5a, 5b) having certain enantiomeric purity (e.g., as describedherein) can be obtained from racemic mixtures by techniques known in theart. Examples include, but are not limited to, the formation of chiralsalts and the use of chiral or high performance liquid chromatography“HPLC” and the formation and crystallization of chiral salts. See, e.g.,Jacques, J., et al., Enantiomers, Racemates and Resolutions(Wiley-Interscience, New York, 1981); Wilen, S. H., et al., Tetrahedron33:2725 (1977); Eliel, E. L., Stereochemistry of Carbon Compounds(McGraw-Hill, NY, 1962); and Wilen, S. H., Tables of Resolving Agentsand Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre DamePress, Notre Dame, Ind., 1972).

Oxidized lipids of the invention (e.g., Formulae 1a, 1b, 2a, 2b, 3a, 3b,4a, 4b, 5a, 5b) having certain enantiomeric purity (e.g., as describedherein) can also be synthesized by the general Methods A or B describedherein using a starting material, or any synthetic intermediate, havingcertain enantiomeric purity, for example,(S)-1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycerol,(S)-1-hexadecyl-2-(4′-carboxymethyl)butyl-glycerol, or(S)-1-eicosanyl-2-(4′-carboxymethyl)butyl-glycerol. The startingmaterial, or any synthetic intermediate, having certain enantiomericpurity can be obtained by synthetic methods known in the art or bychiral separation from racemic mixtures as described herein.

In other embodiments, the present invention relates to a compound madeby any of the methods of synthesis of the invention.

Pharmaceutical Compositions

Other embodiments of the invention relate to a pharmaceuticalcomposition comprising an oxidized lipid of the invention. In someembodiments, the pharmaceutical composition comprises an oxidized lipidof the invention and a pharmaceutically acceptable vehicle. In otherembodiments, the pharmaceutical composition comprises a therapeuticallyeffective amount of the oxidized lipid. In some embodiments, thepharmaceutical composition comprises a therapeutically effective amountof the oxidized lipid and a pharmaceutically acceptable vehicle. As usedherein, a therapeutically effective amount of an oxidized lipid is anamount effective to treat or prevent a disease or disorder of thepresent invention.

In other embodiments, the pharmaceutical compositions of the presentinvention can be orally administered.

In some embodiments, the pharmaceutical composition comprises a compoundhaving a structure according to any of Formulae 1, 2, 3, 4, or 5 asdescribed herein.

In other embodiments, the pharmaceutical composition comprises acompound having a structure of:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof.

In some embodiments, the pharmaceutical composition comprises a compoundselected from the group consisting of(R)-1-hexadecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-701);(R)-1-eicosanyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-702);(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-703);(R)-1-hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-704); and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoricacid pyridiniumethyl ester (VB-705). The prefix “(R)-” refers to theconfiguration of the C-2 carbon of the glycerol backbone. In someembodiments, the compound has an enantiomeric purity of about 80% ee ormore, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee,about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee,about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more. Inother embodiments, the compound has an enantiomeric purity of from about80% ee to about 100% ee, about 85% ee to about 100% ee, about 90% ee toabout 100% ee, about 95% ee to about 100%, about 80% ee to about 99.5%ee, about 85% ee to about 99.5% ee, about 90% ee to about 99.5% ee,about 95% ee to about 99.5%, or any range thereof.

In some embodiments, the pharmaceutical composition comprises a compoundhaving a structure of:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof.

In some embodiments, the pharmaceutical composition comprises a compoundselected from the group consisting of(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-fluoro-pyridiniumethyl ester (VB-706) and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-phenyl-pyridiniumethyl ester (VB-707). The prefix “(R)-” refers to theconfiguration of the C-2 carbon of the glycerol backbone. In someembodiments, the compound has an enantiomeric purity of about 80% ee ormore, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee,about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee,about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more. Inother embodiments, the compound has an enantiomeric purity of from about80% ee to about 100% ee, about 85% ee to about 100% ee, about 90% ee toabout 100% ee, about 95% ee to about 100%, about 80% ee to about 99.5%ee, about 85% ee to about 99.5% ee, about 90% ee to about 99.5% ee,about 95% ee to about 99.5%, or any range thereof.

In other embodiments, the pharmaceutical composition treats or preventsfibrosis (e.g., liver fibrosis, kidney fibrosis, focal and segmentalglomerulosclerosis, or any other fibrosis described herein) as well as,or better than, telmisartan. In other embodiments, the pharmaceuticalcomposition reduces liver inflammation as well as, or better than,telmisartan. In other embodiments, the pharmaceutical compositionreduces liver fibrosis as well as, or better than, telmisartan. In otherembodiments, the pharmaceutical composition treats or prevents kidneyfibrosis as well as, or better than, telmisartan. In other embodiments,the pharmaceutical composition treats or prevents focal and segmentalglomerulosclerosis as well as, or better than, telmisartan.

In other embodiments, the pharmaceutical composition inhibits formationof ligand-induced phosphorylation of IKK, ERK, AKT or p38 comparable toor more than VB-201(1-hexadecyl-2-(4′-carboxy)butyl-glycero-3-phosphocholine or1-hexadecyl-2-(4′-carboxybutyl)-glycerol-3-phosphocholine)) inhibitsformation of ligand-induced phosphorylation of IKK, ERK, AKT or p38. Inother embodiments, the pharmaceutical composition inhibitsligand-induced cell migration comparable to or more than VB-201 inhibitsligand-induced cell migration. In other embodiments, the ligand is LPS,PGN, PAM3, or MCP1. In other embodiments, the cell migration is monocytemigration.

Methods of Treating or Preventing Fibrosis or Inflammatory Diseases orDisorders

Embodiments of the invention relate to a method for treating orpreventing fibrosis or an inflammatory disease or disorder comprisingadministering an oxidized lipid of the invention. In other embodiments,the invention relates to a method for treating or preventing aninflammatory disease or disorder. In other embodiments, the methodcomprises administering a therapeutically effective amount of anoxidized lipid of the invention to a subject in need thereof. In otherembodiments, the method comprises administering a pharmaceuticalcomposition of the invention.

The methods described herein can be used for treating or preventing alltypes fibrosis. In some embodiments of the methods of the invention, thefibrosis is pulmonary fibrosis, liver fibrosis, skin fibrosis or kidneyfibrosis. In some embodiments of the methods of the invention, thefibrosis is heart fibrosis, bone marrow fibrosis, intestine fibrosis,joint fibrosis (knee, shoulder, or other joints), hand fibrosis, fingerfibrosis, skeletal muscle fibrosis, neurofibrosis, and penis fibrosis.In other embodiments, the fibrosis is idiopathic pulmonary fibrosis(IPF), cystic fibrosis, progressive massive fibrosis, cirrhosis,steatohepatitis (fatty liver disease), nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), endomyocardial fibrosis,myocardial infarction, atrial fibrosis, medastinal fibrosis,myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis,keloid, Crohn's disease, scleroderma/systemic sclerosis, arthrofibrosis,Peyronie's disease, Dupuytren's contracture, adhesive capsulitis, orfocal and segmental glomerulosclerosis. In some embodiments, thefibrosis is liver fibrosis. In some embodiments, the fibrosis is kidneyfibrosis. In some embodiments, the subject in need of treatment orprevention of kidney fibrosis has a chronic kidney disease. In someembodiments, the fibrosis is focal and segmental glomerulosclerosis. Insome embodiments, the subject in need of treatment or prevention offocal and segmental glomerulosclerosis has a chronic kidney disease.

In some embodiments, the fibrosis is a fibrosis that does not includeidiopathic pulmonary fibrosis. In other embodiments, the fibrosis is afibrosis that does not include cystic fibrosis. In other embodiments,the fibrosis is a fibrosis that does not include progressive massivefibrosis. In some embodiments, the fibrosis is a fibrosis that does notinclude cirrhosis. In some embodiments, the fibrosis is a fibrosis thatdoes not include steatohepatitis (fatty liver disease). In someembodiments, the fibrosis is a fibrosis that does not includenonalcoholic fatty liver disease (NAFLD). In some embodiments, thefibrosis is a fibrosis that does not include nonalcoholicsteatohepatitis (NASH). In some embodiments, the fibrosis is a fibrosisthat does not include endomyocardial fibrosis. In some embodiments, thefibrosis is a fibrosis that does not include myocardial infarction. Insome embodiments, the fibrosis is a fibrosis that does not includeatrial fibrosis. In some embodiments, the fibrosis is a fibrosis thatdoes not include medastinal fibrosis. In some embodiments, the fibrosisis a fibrosis that does not include myelofibrosis. In some embodiments,the fibrosis is a fibrosis that does not include retroperitonealfibrosis. In some embodiments, the fibrosis is a fibrosis that does notinclude nephrogenic systemic fibrosis. In some embodiments, the fibrosisis a fibrosis that does not include keloid. In some embodiments, thefibrosis is a fibrosis that does not include Crohn's disease. In someembodiments, the fibrosis is a fibrosis that does not includescleroderma/systemic sclerosis. In some embodiments, the fibrosis is afibrosis that does not include arthrofibrosis. In some embodiments, thefibrosis is a fibrosis that does not include Peyronie's disease. In someembodiments, the fibrosis is a fibrosis that does not includeDupuytren's contracture. In some embodiments, the fibrosis is a fibrosisthat does not include adhesive capsulitis. In some embodiments, thefibrosis is a fibrosis that does not include focal and segmentalglomerulosclerosis. In some embodiments, the fibrosis is a fibrosis thatdoes not include fibrous lesions or plaques in the arteries.

In some embodiments, the oxidized lipid treats or prevents liverinflammation, but does not alter liver fibrosis. In other embodiments,the oxidized lipid treats or prevents liver fibrosis, but does not alterliver inflammation.

In other embodiments, the inflammatory disease or disorder is liverinflammation, atherosclerosis, rheumatoid arthritis, inflammatory boweldisease, ulcerative colitis, multiple sclerosis, or psoriasis.

In some embodiments, the inflammatory disease or disorder is aninflammatory cardiovascular disease or disorder, a cerebrovasculardisease or disorder, or a peripheral vascular disease or disorder.

In some embodiments, the inflammatory disease or disorder is aninflammatory cardiovascular disease or disorder selected from the groupconsisting of occlusive diseases or disorders, atherosclerosis, cardiacvalvular disease, stenosis, restenosis, in-stent-stenosis, myocardialinfarction, coronary arterial disease, acute coronary syndromes,congestive heart failure, angina pectoris, myocardial ischemia,thrombosis, Wegener's granulomatosis, Takayasu's arteritis, Kawasakisyndrome, anti-factor VIII autoimmune diseases or disorders, necrotizingsmall vessel vasculitis, microscopic polyangiitis, Churg and Strausssyndrome, pauci-immune focal necrotizing glomerulonephritis, crescenticglomerulonephritis, antiphospholipid syndrome, antibody induced heartfailure, thrombocytopenic purpura, autoimmune hemolytic anemia, cardiacautoimmunity, Chagas' disease or disorder, and anti-helper T lymphocyteautoimmunity.

In some embodiments, the inflammatory disease or disorder is acerebrovascular disease or disorder selected from the group consistingof stroke, cerebrovascular inflammation, cerebral hemorrhage, andvertebral arterial insufficiency.

In some embodiments, the inflammatory disease or disorder is aperipheral vascular disease or disorder is selected from the groupconsisting of gangrene, diabetic vasculopathy, ischemic bowel disease,thrombosis, diabetic retinopathy, and diabetic nephropathy.

In other embodiments, activity of TLR2, TLR4 and/or CD14 is inhibited ina treated cell. In some embodiments, activity of TLR2 and TLR4 isinhibited; activity of TLR4 and CD14 is inhibited; activity of TLR2 andCD14 is inhibited; or activity of TLR2, TLR4 and CD14 is inhibited.

In other embodiments, steatosis in a subject treated with an oxidizedlipid of the invention is not reduced, compared to that in untreated orplacebo-treated subjects. In other embodiments, liver lobular formationin a subject treated with an oxidized lipid of the invention isdecreased, compared to that in untreated or placebo-treated subjects. Inother embodiments, liver lobular formulation in a subject treated withan oxidized lipid of the invention is not decreased, compared to that inuntreated or placebo-treated subjects. In other embodiments, steatosisin a subject treated with an oxidized lipid of the invention is notreduced and liver lobular formation in a subject treated with anoxidized lipid of the invention is decreased, compared to those inuntreated or placebo-treated subjects, respectively. In otherembodiments, steatosis in a subject treated with an oxidized lipid ofthe invention is not reduced and liver lobular formation in a subjecttreated with an oxidized lipid of the invention is not decreased,compared to those in untreated or placebo-treated subjects,respectively. In other embodiments, foam cell-like macrophages aredecreased in a subject treated with an oxidized lipid of the invention,compared to that in untreated or placebo-treated subjects. In someembodiments, liver lobular formation and foam cell-like macrophages in asubject treated with an oxidized lipid of the invention are decreased,compared to those in untreated or placebo-treated subjects,respectively. In some embodiments, liver lobular inflammation in asubject treated with an oxidized lipid of the invention is decreased,compared to that in untreated or placebo-treated subjects. In someembodiments, liver lobular inflammation and foam cell-like macrophagesin a subject treated with an oxidized lipid of the invention aredecreased, compared to those in untreated or placebo-treated subjects,respectively. In some embodiments, liver lobular formation, liverlobular inflammation and foam cell-like macrophages in a subject treatedwith an oxidized lipid of the invention are decreased, compared to thosein untreated or placebo-treated subjects, respectively. In someembodiments, liver lobular formation in a subject treated with anoxidized lipid of the invention is decreased by about 5% to about 50%(e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%,or any ranges between the specified values) compared to that inuntreated or placebo-treated subjects. In some embodiments, theformation of foam cell-like macrophages in a subject treated with anoxidized lipid of the invention is decreased by about 5% to about 50%(e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%,or any ranges between the specified values) compared to that inuntreated or placebo-treated subjects. In some embodiments, liverlobular inflammation in a subject treated with an oxidized lipid of theinvention is decreased by about 5% to about 50% (e.g., about 5%, about10%, about 20%, about 30%, about 40%, about 50%, or any ranges betweenthe specified values) compared to that in untreated or placebo-treatedsubjects.

In some embodiments, the oxidized lipid is a compound having a structureaccording to any of Formulae 1, 2, 3, 4, or 5 as described herein.

In some embodiments, the oxidized lipid is a compound having a structureof:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof.

In some embodiments, the oxidized lipid is a compound selected from thegroup consisting of(R)-1-hexadecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-701);(R)-1-eicosanyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-702);(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-703);(R)-1-hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-704); and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoricacid pyridiniumethyl ester (VB-705). In some embodiments, the compoundhas an enantiomeric purity of about 80% ee or more, e.g., about 80% ee,about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee,about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee,about 99% ee, about 99.5% ee or more. In other embodiments, the compoundhas an enantiomeric purity of from about 80% ee to about 100% ee, about85% ee to about 100% ee, about 90% ee to about 100% ee, about 95% ee toabout 100%, about 80% ee to about 99.5% ee, about 85% ee to about 99.5%ee, about 90% ee to about 99.5% ee, about 95% ee to about 99.5%, or anyrange thereof.

In some embodiments, the oxidized lipid is a compound having a structureof:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof.

In some embodiments, the oxidized lipid is a compound selected from thegroup consisting of(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-fluoro-pyridiniumethyl ester (VB-706) and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-phenyl-pyridiniumethyl ester (VB-707). The prefix “(R)-” refers to theconfiguration of the C-2 carbon of the glycerol backbone. In someembodiments, the compound has an enantiomeric purity of about 80% ee ormore, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee,about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee,about 9′7% ee, about 98% ee, about 99% ee, about 99.5% ee or more. Inother embodiments, the compound has an enantiomeric purity of from about80% ee to about 100% ee, about 85% ee to about 100% ee, about 90% ee toabout 100% ee, about 95% ee to about 100%, about 80% ee to about 99.5%ee, about 85% ee to about 99.5% ee, about 90% ee to about 99.5% ee,about 95% ee to about 99.5%, or any range thereof.

In other embodiments, the oxidized lipid compound treats or preventsfibrosis (e.g., liver fibrosis, kidney fibrosis, focal and segmentalglomerulosclerosis, or any other fibrosis described herein) as well as,or better than, telmisartan. In other embodiments, the oxidized lipidcompound reduces liver inflammation as well as, or better than,telmisartan. In other embodiments, the oxidized lipid compound reducesliver fibrosis as well as, or better than, telmisartan. In otherembodiments, an oxidized lipid compound of the invention treats orprevents kidney fibrosis as well as, or better than, telmisartan. Inother embodiments, an oxidized lipid compound of the invention treats orprevents focal and segmental glomerulosclerosis as well as, or betterthan, telmisartan.

In other embodiments, the oxidized lipid compound inhibits formation ofligand-induced phosphorylation of IKK, ERK, AKT, or p38 comparable to ormore than VB-201(1-hexadecyl-2-(4′-carboxy)butyl-glycero-3-phosphocholine or1-hexadecyl-2-(4′-carboxybutyl)-glycerol-3-phosphocholine) inhibitsformation of ligand-induced phosphorylation of IKK, ERK, AKT, or p38. Inother embodiments, the compound inhibits ligand-induced cell migrationcomparable to or more than VB-201 inhibits ligand-induced cellmigration. In other embodiments, the ligand is LPS, PGN, MCP1, or PAM3.In other embodiments, the cell migration is monocyte migration.

In other embodiments, the subject is a mammal or a human. In otherembodiments, the human is a female. In other embodiments, the human is amale.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 6:

or a stereoisomer, a stereoisomeric mixture, or a salt thereof,

wherein:

n is an integer from 1 to 6, wherein when n is 1, Cn, Bn, Rn, and Y areabsent, and C₁ is attached to R′n;

each of B₁, B₂, . . . Bn−1 and Bn is independently selected from thegroup consisting of oxygen, sulfur, nitrogen, phosphorus and silicon,wherein each of said nitrogen, phosphorus and silicon is optionallysubstituted by one or more substituents selected from the groupconsisting of alkyl, halo, cycloalkyl, aryl, hydroxy, thiohydroxy,alkoxy, aryloxy, thioaryloxy, thioalkoxy, and oxo;

each of A₁, A₂, . . . An−1 and An is independently selected from thegroup consisting of CR″R′″, C═O and C═S,

Y is a moiety having the general formula:

wherein:

each of U, B′ and B″ is independently selected from the group consistingof sulfur and oxygen; and

each of D′ and D″ is independently selected from the group consisting ofhydrogen, a negative charge, alkyl, amino substituted alkyl,heteroalicyclic substituted alkyl, heteroaryl substituted alkyl,cycloalkyl, phosphonate, and thiophosphonate; and

each of X₁, X₂, . . . Xn−1 is independently a saturated or unsaturatedhydrocarbon having the general Formula 7:

wherein m is an integer from 1 to 26; and

Z is selected from the group consisting of:

-   -   H,

-   -    and —OR″,

wherein W is selected from the group consisting of oxygen and sulfur;

wherein at least one of X₁, X₂, . . . Xn−1 comprises a Z other thanhydrogen,

and wherein:

each of R₁, R′₁, R₂, . . . Rn−1, Rn, R′n, each of R″ and R′″ and each ofRa, R′a, Rb, R′b, . . . Rm−1, R′m−1, Rm and R′m is independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl, halo, trihalomethyl, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, phosphonate, phosphate,phosphinyl, sulfonyl, sulfinyl, sulfonamide, amide, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, C-carbamate, N-carbamate,C-thiocarboxy, S-thiocarboxy and amino, or, alternatively, at least twoof R₁, R′₁, R2, . . . Rn−1, Rn and R′n and/or at least two of Ra, R′a,Rb, . . . Rm−1, R′m−1, Rm and R′m form at least one four-, five- orsix-membered aromatic, heteroaromatic, alicyclic or heteroalicyclicring.

In some embodiments, an oxidized lipid of the invention is a compoundhaving a structure according to Formula 6 as defined herein, or astereoisomer, a stereoisomeric mixture, or a salt thereof, wherein Y is

wherein:

n is 3;

each of U, B′ and B″ is oxygen; and

wherein one of D′ and D″ is a heteroalicyclic substituted alkyl orheteroaryl substituted alkyl, and the other of D′ and D″ is hydrogen ora negative charge. In some embodiments, one of D′ and D″ is a heteroarylsubstituted alkyl, and the other of D′ and D″ is hydrogen or a negativecharge.

In some embodiments, the invention relates to deuterated analogs,prodrugs, hydrates, and solvates of any of the oxidized lipid describedherein (e.g., having a structure according to Formulae 1-6).

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in anon-limiting fashion.

Example 1 General Synthetic Procedures

Procedure A

1-alkyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoric acidbromoethyl ester

1-alkyl-2-(4′-carboxymethyl)butyl-glycerol was dried by azeotropicdistillation from benzene. After cooling to room temperature undernitrogen, (2-bromoethyl)-phosphorylchloride (1.5 eq) was added and themixture resulted was stirred at room temperature for 15 minutes. Thereaction mixture was then cooled in an ice bath. Pyridine (anhydrous,1.2 eq) was then added dropwise. The reaction mixture was then allowedto stir at room temperature overnight. After which, the solvent wasremoved under reduced pressure. Water was then added and the reactionmixture was refluxed for 1 hr. After cooling to room temperature, thereaction mixture was extracted with ether. The combined organic phasewas washed with water, dried over sodium sulfate, and then concentratedunder reduced pressure to yield1-alkyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoric acidbromoethyl ester.

1-alkyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid pyridiniumethylester

1-alkyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoric acidbromoethyl ester was dissolved in pyridine (anhydrous). The reactionmixture resulted was refluxed for 22 hrs. After removal of the pyridineunder reduced pressure, the residue was dissolved in methanol. Sodiumbicarbonate (4.5 eq) was then added. The resulting mixture was refluxedfor 1 hr and hot filtered. The residue obtained after washing of thefiltrate with methanol and removal of solvent under reduced pressure waspurified by chromatography on a column of silica gel. Elution withmixtures of CHCl₃:MeOH (5-20%, v/v), followed by CHCl₃:MeOH:H₂O(70:26:4, v/v/v).

Procedure B

O-tosyl ethylene glycol

A solution of p-toluenesulfonyl chloride (7 g) in dichloromethane(anhydrous, 50 ml) was added dropwise at 0° C. to a stirred solution ofethylene glycol (anhydrous, 20 ml) in dichloromethane (anhydrous, 100ml) and pyridine (anhydrous, 7 ml). The resulting mixture was stirred atroom temperature overnight and then poured on ice and allowed to reachroom temperature. The reaction mixture was extracted withdichloromethane (3×100 ml) and the combined organic phase was washedsequentially with water (150 ml), sulfuric acid (2%, 100 ml), water (150ml), saturated sodium bicarbonate (150 ml) and again with water (150ml). Removing the solvent under reduced pressure yielded O-tosylethylene glycol 7.74 g as a colorless oil.

1-alkyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoric acidtosylethyl ester

To a solution of 1-alkyl-2-(4′-carboxymethyl)butyl-glycerol (dried byazeotropic distillation with benzene) and triethylamine (3 eq) in dryTHF, an ice cooled solution of Phosphorus oxychloride (1.2 eq) in dryTHF was added dropwise (over 2 hrs). The reaction mixture was stirred at0° C. for an additional 15 minutes and then at room temperature of 45minutes. After which, the reaction mixture was cooled again to 0° C. Asolution of O-tosyl ethylene glycol (1.1 eq, dried by azeotropicdistillation from benzene) in dry THF was added dropwise over 30 min.The resulting mixture was stirred at room temperature for overnight, andthen filtered. The solvent was removed under reduced pressure. Theresidue obtained was dissolved in water and was refluxed for 1 hr. Afterbeing cooled to room temperature, the reaction mixture was extractedwith ether. The combined organic phase was washed with water andconcentrated under reduced pressure. The residue resulted was purifiedby chromatography on silica gel. Elution with a mixture of CHCl₃:MeOH(5-20%, v/v), followed by removal of solvent under reduced pressureyielded 1-alky-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoric acidtosylethyl ester.

1-alkyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid pyridiniumethylester

A solution of 1-alkyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoricacid tosylethyl ester in pyridine was stirred and heated to 40° C. for 6hrs. The solution was then cooled and stirred at room temperatureovernight. After removing the solvent under reduced pressure, theresidue obtained was purified by chromatography on silica gel. A mixtureof CHCl₃:MeOH (5-20%, v/v) was used as initial eluent, which wasfollowed by a mixture of CHCl₃:MeOH:H₂O (70:26:4, v/v/v). The fractionscollected were concentrated under reduced pressure. The residue obtainedwas then dissolved in chloroform, which was dried over sodium sulfate.Removal of chloroform under reduced pressure then yielded purified1-alkyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid pyridiniumethylester.

The general procedures above are applicable for synthesis of theoxidized lipid described herein. For methyl ester methylation, there isan additional step in the procedure: methylation in methanol and HCl.

General Procedure for Methyl Ester Formation

1-alkyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid pyridiniumethylester was dissolved in methanol. Hydrochloric acid was added to themethanol solution and the mixture stirred at room temperature for 4 hrs.After which, water was added to the reaction mixture. The resultingmixture was extracted with CHCl₃. The organic phase was washedsequentially with water, saturated sodium bicarbonate and again withwater, and then dried over sodium sulfate. Removal of the solvent underreduced pressure then yielded1-alkyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoric acidpyridiniumethyl ester.

Example 2 Synthesis of VB-703

VB-703 was synthesized by two synthetic procedures according to thegeneral procedures described above.

Procedure A

1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoricacid bromoethyl ester

1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycerol (13.75 g) wasdissolved in benzene (100 ml) and dried by azeotropic distillation of 50ml benzene. After cooling to room temperature under nitrogen,(2-bromoethyl)-phosphorylchloride (5.43 ml, 10.25 g) was added. Theresulting mixture was stirred at room temperature for 15 minutes andthen cooled to 0° C. Pyridine (anhydrous, 2.73 ml, 2.68 g) was addeddropwise. The reaction mixture was stirred at room temperature overnightand then the solvent removed under reduced pressure. Water (100 ml) wasadded and the mixture refluxed for 1 hr. After cooling to roomtemperature, the reaction mixture was extracted with ether (3×100 ml)and washed with water (100 ml). Drying over sodium sulfate and removalof the solvent under reduced pressure yielded 15.06 g of1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoricacid bromoethyl ester.

1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acidpyridiniumethyl ester

1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-3-phosphoricacid bromoethyl ester (15.06 g) was dissolved in pyridine (anhydrous, 50ml) and the reaction mixture refluxed for 22 hrs. After removal of thesolvent under reduced pressure, the residue was dissolved in methanol(100 ml) and sodium bicarbonate (10.68 g) was added. The resultingsolution was refluxed for 1 hour (hr) and hot filtered. The residueobtained after washing of the filtrate with methanol (2×15 ml) andremoval of solvent under reduced pressure was purified by chromatographyon a column of silica gel (213 g). Elution with mixtures of CHCl₃:MeOH(5-20%, v/v) followed by CHCl₃:MeOH:H₂O (70:26:4, v/v/v). The solventwas removed under reduced pressure and the residue dissolved inchloroform. Drying of the solution over sodium sulfate, filtration andremoval of the solvent under reduced pressure yielded 2.42 g1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acidpyridiniumethyl ester.

Procedure B

1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoricacid tosylethyl ester

A solution of 1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycerol(7.47 g, dried by azeotropic distillation with benzene) andtriethylamine (7 ml) in dry THF (80 ml) was added dropwise for 2 hrs at0° C. to a stirred solution of phosphorus oxychloride (2 ml) in dry THF(50 ml). The reaction mixture stirred at 0° C. for an additional 15minutes and at room temperature for an additional 45 minutes. Thereaction mixture was cooled again to 0° C. and a solution of O-tosylethylene glycol (3.65 g, dried by azeotropic distillation with benzene)in dry THF (50 ml) was added dropwise for 30 min. The resulting mixturewas stirred at room temperature overnight, then filtered, and thesolvent removed under reduced pressure. The residue was dissolved inwater (100 ml), refluxed for 1 hr, then cooled and extracted with ether(2×100 ml). The combined organic phase was washed with water (100 ml)and the solvent removed under reduced pressure. The residue (9.62 g) waspurified by chromatography on a column of silica gel (250 g). Elutionwith mixtures of CHCl₃:MeOH (5-20%, v/v) and removal of solvent underreduced pressure yielded 6.90 g of1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoricacid tosylethyl ester.

1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acidpyridiniumethyl ester

A solution of1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoricacid tosylethyl ester (2.20 g) in pyridine (30 ml) was stirred andheated to 40° C. for 6 hrs, then cooled and stirred at room temperatureovernight. After removing the solvent under reduced pressure, theresidue was purified by chromatography on column of silica gel (95 g).Elution was done with mixtures of CHCl₃:MeOH (5-20%, v/v) followed byCHCl₃:MeOH:H2O (70:26:4, v/v/v). The solvent was removed under reducedpressure and the residue dissolved in chloroform. Drying of the solutionover sodium sulfate, filtration and removal of the solvent under reducedpressure yielded 0.4 g of1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acidtosylethyl ester.

Mass spectrometry (MS): Calculated for C₃₅H₆₄NO₈P 657. Found (ESI+) 658[M+H]⁺, 680 [M+Na]⁺ and 696 [M+K]⁺.

¹H-NMR (700 MHz, CDCl₃, TMS ref): δ 0.88 (t, 6H, 2 CH3), 1.24-1.29 (m,32H, 16 CH2), 1.53 (m, 1H, CH), 1.59 (tt, 2H, CH2), 1.69 (m, 1H, CH2),2.34 (t, 2H, CH2), 3.29 (dt, 2H, CH2), 3.41 (m, 1H, CH2), 3.46 (m, 1H,CH2), 3.55 (m, 1H, CH2), 3.60 (m, 1H, CH), 3.66 (m, 1H, CH2), 3.77 (m,1H, CH2), 3.88 (br s, 1H, CH2), 4.33 ((br, 2H, CH2), 4.87 ((br, 2H,CH2), 8.07 (t, 2H, meta-aromatic), 8.49 (t, 1H, para-aromatic), 9.03 (d,2H, ortho-aromatic).

¹³C-NMR: δ 14.17, 22.07, 22.85, 26.99, 27.01, 29.50, 29.54, 29.83,29.84, 29.88, 30.29, 31.42, 31.46, 32.11, 34.09, 38.35, 62.41, 63.86,65.99, 70.20, 71.16, 75.20, 78.29 (CH), 128.35 (meta aromatic), 145.66(para aromatic), 145.75 (ortho aromatic), 176.67 (CO2H).

Example 3 Synthesis of VB-701

1-hexadecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acidpyridiniumethyl ester (VB-701) was synthesized by following the generalprocedure A as described in Example 1. Mass spectrometry (MS):Calculated for C₃₁H₅₆NO₈P 601 Found (ESI+) 602 [M+H]⁺ and 624 [M+Na]⁺.

Example 4 Synthesis of VB-702

1-eicosanyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acidpyridiniumethyl ester (VB-702) was synthesized by following the generalprocedure A as described in Example 1. Mass spectrometry (MS):Calculated for C₃₅H₆₄NO₈P 657.4. Found (ESI+) 658.5 [M+H]⁺ and 680.5[M+Na]⁺.

¹H-NMR (700 MHz, CDCl₃, TMS ref): δ 089 (t, 3H, CH3), 1.27-1.32 (m, 34H,17 CH2), 1.54 (tt, 2H, CH2), 1.60 (m, 2H, CH2), 1.66 (m, 1H, CH2), 1.73(m, 1H, CH2), 2.31 (br, t, 2H, CH2), 3.41 (m, 1H, CH2), 3.42 (m, 1H,CH2), 3.47 (m, 1H, CH2), 3.56 (m, 1H, CH), 3.58 (m, 1H, CH2), 3.65 (m,1H, CH2), 3.80 (m, 1H, CH2), 3.88 (m, 1H, CH2), 4.38 ((br, 2H, CH2),4.95 ((br, 2H, CH2), 8.10 (t, 2H, aromatic), 8.52 (t, 1H, aromatic),9.10 (br, 2H, aromatic). ¹³C-NMR: δ 14.22, 22.41, 22.99, 29.68, 29.85,29.98-30.02, 32.26, 35.24, 62.51, 64.24, 66.02, 70.39, 70.56, 72.17,78.27 (CH), 128.47 (aromatic), 145.72 (aromatic), 146.09 (aromatic),179.08 (CO2H).

Example 5 Synthesis of VB-704

1-hexadecyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoric acidpyridiniumethyl ester (VB-704) was synthesized by following the generalprocedure for methyl ester formation as described in Example 1. Massspectrometry (MS): Calculated for C₃₂H₅₈NO₈P 615; Found (ESI+) 616[M+H]⁺ and 638 [M+Na]⁺.

Example 6 Synthesis of VB-705

1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-glycero-sn-3-phosphoricacid pyridiniumethyl ester (VB-705)_was synthesized by following thegeneral procedure for methyl ester formation as described in Example 1.Mass spectrometry (MS): Calculated for C₃₆H₆₆NO₈P 671; Found (ESI+) 672[M+H]⁺ and 694 [M+Na]⁺.

Example 7 Synthesis of VB-706

1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-fluoro-pyridiniumethyl ester (VB-706)_was synthesized by following thegeneral procedure B as described in Example 1. Mass spectrometry (MS):Calculated for C₃₅H₆₃FNO₈P 675.43. Found (ESI+) 676.58 [M+H]⁺ and 698.57[M+Na]⁺.

¹H-NMR (700 MHz, CDCl₃, TMS ref.): δ 088 (t, 6H, 2 CH3), 1.26-1.30 (m,32H, 16 CH2), 1.53 (m, 1H, CH), 1.60 (tt, 2H, CH2), 1.69 (m, 1H, CH2),2.31 (t, 2H, CH2), 3.30 (dt, 2H, CH2),3.42 (m, 1H, CH2), 3.46 (m, 1H,CH2), (3.55 (m, 1H, CH2), 3.60 (m, 1H, CH2), 3.68 (m, 1H, CH2), 3.76 (m,1H, CH2), 3.89 (m, 1H, CH2), 4.31 (br, 2H, CH2), 4.88 ((br, 2H, CH2),8.14 (br, 1H, meta-aromatic), 8.35 (t, 1H, para-aromatic), 8.96 (d, 1H,ortho-aromatic), 9.25 (s, 1H, ortho-aromatic).

¹³C-NMR: δ 14.18, 22.39, 22.88, 27.02, 27.04, 29.50-30.30, 31.44, 31.48,32.14, 35.00, 38.37, 63.08, 63.66, 66.08, 70.30, 75.24, 78.37 (CH),129.65 (meta aromatic), 133.36 (para aromatic), 135.79 (ortho-aromatic),143.05 (ortho aromatic), 160.81 (CF), 178.16 (CO2H).

¹⁹F NMR (376.4 MHz)-111.55.

Example 8 Synthesis of VB-707

1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-phenyl-pyridiniumethyl ester (VB-707)_was synthesized by following thegeneral procedure B as described in Example 1. Mass spectrometry (MS):Calculated for C₄₁H₆₈NO₈P 733. Found (ESI+) 734 [M+H]⁺ and 756 [M+Na]⁺.

Example 9 VB-701 Inhibits LPS (TLR4)-Induced Signaling in HumanMonocytes (Primary CD14+)

Methods and Materials

Isolation of Monocytes

Venous blood samples were obtained from healthy male donors incompliance with the Institutional Review Board at the Sheba MedicalCenter, Ramat Gan, Israel. PBMCs were isolated on Ficoll-Paque PLUS (GEHealthcare, Uppsala, Sweden) using 50 ml Leucosep tubes (GreinerBio-One, Frickenhausen, Germany). Cells were washed in PBS (Kibbutz BeitHaemek, Israel) and incubated at 4° C. for 15 minutes in a buffercontaining PBS and 0.5% bovine serum albumin (BSA) with human CD14microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

Activation of Cells and Western Blotting

Cells (10⁶/ml) were pretreated for 20 min with VB-201(1-hexadecyl-2-(4′-carboxy)butyl-glycero-3-phosphocholine or1-hexadecyl-2-(4′-carboxybutyl)-glycerol-3-phosphocholine) or VB-701 atthe doses indicated in FIG. 2, or with solvent (Sol), followed by 15 minactivation with 100 ng/ml lipopolysaccharide (LPS) or were untreated(Unt). Cells were washed and resuspended in lysis buffer containing1:100 dithiothreitol (DTT), phosphatase and protease inhibitors (ThermoScientific). Samples were loaded onto a precast Criterion TGX gel(Bio-Rad, Hemel Hempstead, UK) and transferred onto nitrocellulosemembrane. Blots were blocked with 5% milk or BSA in Tris buffered salineand Tween 20 (TBST) for 1 h, followed by incubation with primary andsecondary antibodies. Membranes were developed using an ECL kit (ThermoScientific). The following antibodies were used for immunoblotting:

Primary antibodies: p-p38 (Cat. No. 4511; 1:1000) and p-IKK (Cat. No.2697; 1:1000) were from Cell Signaling Technology (Danvers, Mass., USA).p-ERK1/2 (Cat. No. M8159; 1:10000) was purchased from Sigma (Israel).αTubulin (Tub) served as a loading control.

Secondary antibodies: HRP donkey anti-rabbit (1:5000) and HRP goatanti-mouse (1:3000) were from Jackson ImmunoResearch (West Grove, Pa.,USA). HRP donkey anti-goat (1:5000) was from Santa Cruz Biotechnology.

Results

FIG. 2 shows that VB-701 and VB-201 inhibit formation of p-IKK, p-ERKand p-p38 induced by LPS in human monocytes. Accordingly, VB-701 andVB-201 inhibit LPS (TLR4)-induced signaling in human monocytes (primaryCD14+).

Example 10 VB-701 Inhibits PGN (TLR2)-Induced Signaling in HumanMonocytes (THP-1 Cell Line)

Methods and Materials

Activation of Cells and Western Blotting

The monocytic THP-1 cell line was purchased from the American TypeTissue Culture Collection (ATCC Cat. No. TIB-202). Cells (10⁶/ml) werepretreated for 20 min with VB-201 or VB-701 at the doses indicated inFIG. 3, or with solvent, followed by activation with 20 μg/mlpeptidoglycan (PGN) (InvivoGen, San Diego, Calif.) for 15 minutes, orwere untreated (“Unt”). Cells were washed and resuspended in lysisbuffer containing 1:100 dithiothreitol (DTT), phosphatase and proteaseinhibitors (Thermo Scientific). Samples were loaded onto a precastCriterion TGX gel (Bio-Rad, Hemel Hempstead, UK) and transferred ontonitrocellulose membrane. Blots were blocked with 5% milk or BSA in Trisbuffered saline and Tween 20 (TBST) for 1 h, followed by incubation withprimary and secondary antibodies. Membranes were developed using an ECLkit (Thermo Scientific). The following antibodies were used forimmunoblotting:

Primary antibodies: p-p38 (Cat. No. 4511; 1:1000) and p-IKK (Cat. No.2697; 1:1000) were from Cell Signaling Technology (Danvers, Mass., USA).p-ERK1/2 (Cat. No. M8159; 1:10000) was purchased from Sigma (Israel).αTubulin served as a loading control.

Secondary antibodies: HRP donkey anti-rabbit (1:5000) and HRP goatanti-mouse (1:3000) were from Jackson ImmunoResearch (West Grove, Pa.,USA). HRP donkey anti-goat (1:5000) was from Santa Cruz Biotechnology.

Results

FIG. 3 shows that VB-701 and VB-201 inhibit formation of p-IKK, p-ERKand p-p38 induced by PGN in THP-1 cells. Accordingly, VB-701 and VB-201inhibit PGN (TLR2)-induced signaling.

Example 11 VB-701 Inhibits MCP-1-Induced Signaling in Human Monocytes(THP-1 Cell Line)

Methods and Materials

Activation of Cells and Western Blotting

THP-1 cells (10⁶/ml) were pretreated for 20 min with VB-201 or VB-701 atthe doses indicated in FIG. 4, or with solvent, followed by activationwith 50 ng/ml MCP1, or were untreated (“Unt”). Cells were washed andresuspended in lysis buffer containing 1:100 dithiothreitol (DTT),phosphatase and protease inhibitors (Thermo Scientific). Samples wereloaded onto a precast Criterion TGX gel (Bio-Rad, Hemel Hempstead, UK)and transferred onto nitrocellulose membrane. Blots were blocked with 5%milk or BSA in Tris buffered saline and Tween 20 (TBST) for 1 h,followed by incubation with primary and secondary antibodies. Membraneswere developed using an ECL kit (Thermo Scientific). The followingantibodies were used for immunoblotting:

Primary antibodies: p-ERK1/2 (Cat. No. M8159; 1:10000) was purchasedfrom Sigma (Israel). p-AKT (Cat. No. 4060; 1:1000) was purchased fromCell Signaling Technology (Danvers, Mass.). αTubulin served as a loadingcontrol and was purchased from Sigma (Israel).

Secondary antibodies: HRP donkey anti-rabbit (1:5000) and HRP goatanti-mouse (1:3000) were from Jackson ImmunoResearch (West Grove, Pa.,USA). HRP donkey anti-goat (1:5000) was from Santa Cruz Biotechnology.

Results

FIG. 4 shows that VB-701 and VB-201 inhibit formation of p-AKT and p-ERKinduced by MCP1 in THP-1 cells. Accordingly, VB-701 and VB-201 inhibitMCP1-induced signaling.

Example 12 VB-701 Inhibits Chemokine-Induced Migration of HumanMonocytes (Primary CD14+)

Methods and Materials

Isolation of Monocytes

Venous blood samples were obtained from healthy male donors incompliance with the Institutional Review Board at the Sheba MedicalCenter, Ramat Gan, Israel. PBMCs were isolated on Ficoll-Paque PLUS (GEHealthcare, Uppsala, Sweden) using 50 ml Leucosep tubes (GreinerBio-One, Frickenhausen, Germany). Cells were washed in PBS (Kibbutz BeitHaemek, Israel) and incubated at 4° C. for 15 minutes in a buffercontaining PBS and 0.5% bovine serum albumin (BSA) with human CD14microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

Activation of Cells and Cell Migration Trans-well Assay

Cells (10⁶/ml) were pretreated for 20 min with VB-201 or VB-701 at thedoses indicated in FIG. 5, or with solvent (Sol).

To test for chemokine-induced cell migration, RANTES (100 ng/ml; Cat.No. 300-06, PeproTech, Israel) and MCP-1 (50 ng/ml; Cat. No. 300-04,PeproTech, Israel) were dissolved in RPMI-1640 medium supplemented with0.5% fetal bovine serum (FBS) and placed at the lower chamber of QCM24-well, 5 mm pore, migration assay plates (Corning-Costar, Corning,N.Y.). Cells (3×10⁵) were seeded in the upper chamber and incubated for2-4 hours. Subsequently, the number of cells which migrated to the lowercompartment was determined by fluorescence-activated cell sorting(FACS).

Results

FIG. 5 shows that VB-701 and VB-201 inhibit chemokine-induced migrationof human monocytes (primary CD14+).

Example 13 VB-702 Inhibits LPS (TLR4)-Induced Signaling in HumanMonocytes (Primary CD14+)

Human monocytes were obtained, treated and analyzed by western blot asdescribed in Example 9 and FIG. 6. FIG. 6 shows that VB-702 and VB-201inhibit formation of p-IKK, p-ERK and p-p38 induced by LPS in humanmonocytes. Accordingly, VB-702 and VB-201 inhibit LPS (TLR4)-inducedsignaling in human monocytes (primary CD14+).

Example 14 VB-702 Inhibits RANTES-Induced Signaling in Human Monocytes(Primary CD14+)

Human monocytes were obtained, treated and analyzed by western blot asdescribed in Example 9 and FIG. 7, except that cells were induced withRANTES (100 ng/ml; Cat. No. 300-06, PeproTech, Israel) for 15 minutes.FIG. 7 shows that VB-702 and VB-201 inhibit formation of p-ERK inducedby RANTES in human monocytes. Accordingly, VB-702 and VB-201 inhibitRANTES-induced signaling in human monocytes (primary CD14+).

Example 15 VB-702 Inhibits Chemokine-Induced Migration of HumanMonocytes (Primary CD14+)

Human monocytes were obtained, treated and analyzed for cell migrationby trans-well assay as described in Example 12 and FIG. 8. FIG. 8 showsthat VB-702 and VB-201 inhibit chemokine-induced migration of humanmonocytes (primary CD14+).

Example 16 VB-701 and VB-702 Inhibit IL-12p40 Levels in Human Monocytes(Primary CD14+) Stimulated with LPS (via TLR4) or Pam3CSK4(TLR2-Stimulated)

Methods and Materials

Human monocytes were obtained as described in Example 9 and FIG. 9A-9B.Human monocytes were seeded (10⁶/ml) and pretreated for 1 hour withVB-201, VB-701 or VB-702, followed by 24 hour activation with 100 ng/mlLPS from Escherichia coli strain 055:B5 (Sigma, Israel) (FIG. 9A) or 300ng/ml Pam3CSK4 (InvivoGen, San Diego, Calif., USA) (FIG. 9B) to inducecytokine production. IL-12/23p40 concentration in the supernatant wasthen measured by ELISA (R&D systems, Cat. No. DY1240). Cells activatedwith solvent (0.5% ethanol in PBS) were used as a control.

Results

FIGS. 9A-9B show that VB-201, VB-701 and VB-702 inhibit IL-12p40 levelsin in human monocytes (primary CD14+) LPS (TLR4)- and Pam3CSK4(TLR2)-stimulated.

Example 17 VB-703 Inhibits LPS (TLR4)-Induced Signaling, LPS binding,Liver Inflammation and Fibrosis

Methods and Materials

Isolation of Monocytes

Venous blood samples were obtained from healthy male donors incompliance with the Institutional Review Board at the Sheba MedicalCenter, Ramat Gan, Israel. PBMCs were isolated on Ficoll-Paque PLUS (GEHealthcare, Uppsala, Sweden) using 50 ml Leucosep tubes (GreinerBio-One, Frickenhausen, Germany). Cells were washed in PBS (Kibbutz BeitHaemek, Israel) and incubated at 4° C. for 15 minutes in a buffercontaining PBS and 0.5% bovine serum albumin (BSA) with human CD14microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

Activation of Cells and Western Blotting

Cells (10⁶/ml) were pretreated for 20 min with VB-201 or VB-703 followedby 15 min activation with 100 ng/ml LPS. Cells were washed andresuspended in lysis buffer containing 1:100 dithiothreitol (DTT),phosphatase and protease inhibitors (Thermo Scientific). Samples wereloaded onto a precast Criterion TGX gel (Bio-Rad, Hemel Hempstead, UK)and transferred onto nitrocellulose membrane. Blots were blocked with 5%milk or BSA in Tris buffered saline and Tween 20 (TBST) for 1 h,followed by incubation with primary and secondary antibodies. Membraneswere developed using an ECL kit (Thermo Scientific). The followingantibodies were used for immunoblotting:

Primary antibodies: p-p38 (Cat. No. 4511; 1:1000) and p-IKK (Cat. No.2697; 1:1000) were from Cell Signaling Technology (Danvers, Mass., USA).p-ERK1/2 (Cat. No. M8159; 1:10000) was purchased from Sigma (Israel).Heat shock protein (HSP) 90 (Cat. No. 13119; 1:500) was from Santa CruzBiotechnology (Santa Cruz, Calif., USA).

Secondary antibodies: HRP donkey anti-rabbit (1:5000) and HRP goatanti-mouse (1:3000) were from Jackson ImmunoResearch (West Grove, Pa.,USA). HRP donkey anti-goat (1:5000) was from Santa Cruz Biotechnology.

LPS Binding Inhibition Assay

To assess interference with lipopolysaccharide (LPS) binding, VB-201 orVB-703 were incubated for 20 min with cells (10⁶/ml) after which 100ng/ml of biotin-LPS (InvivoGen) was added for an additional 15 minutes,all at 4° C. Cells were washed, resuspended in FACS buffer and analyzedon a FACS-Calibur device.

Induction of NASH and Liver Fibrosis

Neonatal male mice exposed to low-dose streptozotocin (STZ) developliver steatosis with diabetes. Continuous high fat diet (HFD) increaseslobular inflammation with foam cell-like macrophages, showingnonalcoholic steatohepatitis (NASH) pathology. NASH was induced in 40male mice by a single subcutaneous injection of 200 μg per mouse of STZtwo days after birth and feeding HFD [57 kcal % fat]) from four weeks ofage. Vehicle, VB-703 (4 mg/kg), or telmisartan (10 mg/kg) as positivecontrol, were administered once daily for three weeks, starting from sixweeks of age. Mice were sacrificed at nine weeks of age.

Steatohepatitis and Fibrosis Evaluation

Liver pathology was used to determine the effect of VB-703 on liverinflammation and fibrosis. Histology slides were stained withhematoxylin/eosin (H&E) to assess inflammation. The inflammation scorewas determined as follows:

0—no inflammatory foci

1—<2 inflammatory foci

2—2-4 inflammatory foci

3—>4 inflammatory foci

Histology slides were stained with Sirius red to determine collagencontent as a marker for the extent of fibrosis.

Q-PCR

RNA was prepared from livers from normal mice and NASH-induced micetreated with vehicle, VB-703, or telmisartan, using RNeasy mini kit(Qiagen). For cDNA preparation, 2 μg of RNA was combined with qScriptreaction mix and qScript Reverse Transcriptase (Quanta BioSciences) for5 min at 22° C. and then for 30 min at 42° C. Reaction was ended byincubation for an additional 5 min at 85° C. All real time PCR reactionswere performed using the 7300 Real Time PCR System (Applied Biosystems).Q-PCR with mouse ready set of probe with primer was used for IL-1β,IL-12/23p40 and MCP-1 (Applied Biosystems). GAPDH was used to normalizeRNA levels.

Results

Effect of VB-703 and VB-201 on TLR4-Mediated Signaling Events

To determine the effect of VB-201 and VB-703 on TLR4-mediated signalingpathways, isolated human primary monocytes were preincubated with VB-201or VB-703 and then activated with LPS. FIG. 10A shows that VB-703inhibited inflammatory cell signaling molecules phosphorylated ERKkinase (p-ERK), phosphorylated p38 (p-p38) and phosphorylated IKK kinase(p-IKK) in a dose dependent manner. Moreover, VB-703 had at least a10-fold higher efficacy in inhibiting TLR4-driven proteinphosphorylation than VB-201. The effect of VB-703 on the binding of LPSto the TLR4 complex was then tested. FIG. 10B shows that VB-703inhibited the binding of LPS with an IC50 of ˜0.5 m/ml, more than a10-fold lower IC50 than that of VB-201 (˜7 μg/ml).

VB-703 Inhibits Liver Fibrosis

The effect of VB-703 on liver inflammation and fibrosis in a NASH mousemodel was tested. Treatment with VB-703 significantly decreased fibrosisby 58% compared to vehicle treated mice (FIGS. 11A-11B). This effect wasgreater than of the positive control telmisartan (47%). However, VB-703did not appear to significantly alter steatosis (FIGS. 12A-12B).

VB-703 Inhibits Expression of Inflammation Mediators

FIGS. 21A-21C show that the expression of two pro-inflammatorycytokines, IL-10 and IL-12/23p40, and the chemokine MCP-1 weresignificantly inhibited in livers taken from NASH-induced mice that weretreated with VB-703.

Example 18 VB-703 Inhibits PGN (TLR2)-Induced Signaling in HumanMonocytes (THP-1 Cell Line)

THP-1 cells were obtained, treated and analyzed by western blot asdescribed in Example 10 and FIG. 13. FIG. 13 shows that VB-703 andVB-201 inhibit formation of p-IKK, p-ERK and p-p38 induced by PGN THP-1cells. Accordingly, VB-703 and VB-201 inhibit PGN (TLR2)-inducedsignaling in human monocytes (THP-1 cell line).

Example 19 VB-703 Inhibits IL-6 Secretion in LPS (TLR4)-StimulatedMonocyte-Derived Dendritic Cells (Mo-Derived DCs)

Methods and Materials

To generate monocyte-derived DC (Mo-DCs), CD14+ monocytes were counted,washed and seeded (10⁶/ml) in medium containing RPMI-1640, L-glutamine,β-mercaptoethanol, 10% fetal calf serum (FCS), sodium pyruvate,non-essential amino acids, 0.01 M HEPES, antibiotics (penicillin,streptomycin), 50 ng/ml human granulocyte-macrophage colony-stimulatingfactor (GMCSF) and 20 ng/ml human IL-4 (both from PeproTech Asia,Israel). Medium was replaced every 2-3 days. Mo-DCs were collected 5-6days post-culture, counted and seeded (10⁶/ml). Cells were pretreatedfor 1 hour with VB-201 or VB-703, followed by 24 hours activation with100 ng/ml LPS from Escherichia coli strain 055:B5 (Sigma, Israel) toinduce cytokine production. IL-6 concentration (FIG. 14) and IL-12/23p40(FIG. 15) concentration in supernatant were measured by ELISA (R&Dsystems, Cat. No. DY1240 and Cat. No. DY206, respectively). Cellsactivated with solvent (0.5% ethanol in PBS) were used as a control.

Results

FIGS. 14 and 15 show VB-703 and VB-201 inhibit IL-6 (FIG. 14) andIL-12p40 (FIG. 15) secretion in LPS (TLR4) stimulated Mo-Derived DCs.

Example 20 VB-704 Does Not Inhibit LPS-Biotin Binding to Human Monocytes(Primary CD14+)

Human monocytes were obtained, treated and analyzed by LPS bindinginhibition assay as described in Example 17 and FIG. 16. FIG. 16 showsthat VB-704 does not inhibit LPS-biotin binding to human monocytes(primary CD14+).

Example 21 VB-704 Inhibits Chemokine-Induced Migration in HumanMonocytes (primary CD14+)

Human monocytes were obtained, treated and analyzed for cell migrationby trans-well assay as described in Example 12 and FIG. 17. FIG. 17shows that VB-704 inhibits chemokine-induced migration in humanmonocytes (primary CD14+).

Example 22 VB-705 Inhibits TLR4 Mediated Signaling in Human Monocytes(primary CD14+)

Human monocytes were obtained, treated and analyzed by western blot asdescribed in Example 9 and FIG. 18. FIG. 18 shows that VB-705 and VB-201inhibit formation of p-ERK and p-AKT induced by LPS in human monocytes.Accordingly, VB-705 and VB-201 inhibit LPS (TLR4)-induced signaling inhuman monocytes (primary CD14+).

Example 23 VB-705 Inhibits LPS Binding in Human Monocytes (PrimaryCD14+)

Human monocytes were obtained, treated and analyzed with an LPS bindinginhibition assay as described in Example 17 and FIG. 19. FIG. 19 showsthat VB-705 inhibits LPS-biotin binding to human monocytes (primaryCD14+).

Example 24 VB-705 Does Not Inhibit SDF1-Induced Migration of HumanMonocyte (THP-1 Cells)

Methods and Materials

THP-1 cells (10⁶/ml) were pretreated for 20 min with VB-201 or VB-705 at5 μg/ml, or with solvent (Sol). To test for chemokine-induced cellmigration, RANTES (100 ng/ml, Cat. No. 300-06) (PeproTech, Israel) andMCP-1 (50 ng/ml, Cat. No. 300-04) (PeproTech, Israel) were dissolved inRPMI-1640 medium supplemented with 0.5% fetal bovine serum (FBS) andplaced at the lower chamber of QCM 24-well, 5 mm pore, migration assayplates (Corning-Costar, Corning, N.Y.). Cells (3×10⁵) were seeded in theupper chamber and incubated for 2-4 hours. Subsequently, the number ofcells which migrated to the lower compartment was determined byfluorescence-activated cell sorting (FACS).

Results

FIG. 20 shows VB-705 does not inhibit SDF1-induced migration of humanmonocytes (THP-1 cell line).

Example 25 VB-703 and Focal and Segmental Glomerulosclerosis

Methods and Materials

Animals and Experimental Protocol

Male Sprague Dawley (SD) Rats (Harlan Laboratories, Israel) with aninitial weight of 200 g were housed 2-3 per cage in IVC cages indedicated HVAC (heat, ventilation, air conditioning animal facility at atemperature of 22±2° C. and RH (relative humidity) of 55±15%).Temperature and humidity were monitored continuously. The facility hadno exposure to outside light and was maintained on automatic alternatingcycles of 12 hours of light and 12 hours of dark. Animals were providedwith a commercial rodent diet (Harlan Teklad TRM Rat/Mouse Diet) adlibitum and allowed free access to autoclaved water, supplied to eachcage via polysulphone bottles with stainless steel sipper tubes. Allanimal work was approved by the Animal Care and Use Committee of Israel(IL-13-03-027).

Induction of Chronic Renal Disease by 5/6 Nephrectomy

Rats were divided into three groups: (1) Healthy rats (n=3) in group A,(2) Sham group—subjected to chirurgical process but without kidney massreduction (n=3) in group B, and (3) the rest were induced with chronicrenal failure (n=32). Chronic renal failure was induced by a two stage(5/6) nephrectomy (Nx), with subtraction firstly of about ⅔ of the leftkidney by left flank incision and, one week later, complete removal ofthe right kidney. General anesthesia consisted of intraperitonealinjection of ketamine 100 mg/kg and xylazine 20 mg/kg (0.85 mlketamine+0.15 ml xylazine for each ml preparation; 1 μl/g BW wasinjected I.P).

Experimental Groups

One week following the second surgery, rats were randomly assigned tothe following experimental groups:

Healthy, orally administered with vehicle—PBS 0.5% Ethanol (n=3);

Sham-operated, orally administered with vehicle—PBS 0.5% Ethanol (n=3);

Nephrectomized, orally administered with vehicle—PBS 0.5% Ethanol (n=8);

Nephrectomized, orally administered with VB-703 4 mg/kg (n=8); and

Nephrectomized, orally administered with telmisartan 10 mg/kg aspositive control (n=8).

Body weight (BW) was monitored throughout the study and rats weretreated by oral gavage according to their body weight for 7 weeks. Ratswere sacrificed by CO₂ inhalation 8 weeks from removal of the rightkidney (2^(nd) surgery).

Blood Analysis, Albuminuria, and Creatinine Clearance

Proteinuria and albuminuria was determined in urine specimens, collectedduring a 24-hour period, from animals housed in cages at 4 weeks (3 w oftreatment) and 8 weeks (7 w of treatment) after the subtotal nephrectomy(2^(nd) surgery). Urine samples were analyzed for: glucose, urea,sodium, potassium, creatinine, total protein, and albumin.

Serum was collected at 8 weeks after the subtotal nephrectomy (2^(nd)surgery). The serum collected was also analyzed for glucose, urea,sodium, potassium, creatinine, total protein, albumin, and globulin.

Kidney Collection

Upon sacrifice, at 8 weeks, kidneys were collected, weighed and fixed in4% formaldehyde.

Renal Morphology and Morphometric Analysis

For light microscopy, paraffin-embedded tissue slides of 4 μm werestained with Periodic Acid-Schiff (PAS) reagent, Masson's Trichrome andHematoxylin & Eosin.

Glomerular sclerosis index. Glomerulosclerosis was assessed byPAS-stained sections using a semiquantitative scoring system. The extentof glomerulosclerosis was evaluated by examining mostly 100 randomlyselected glomeruli at a magnification of ×400 and applying a scoresystem according to the percentage of sclerosed glomerular area. Thescore was graded from 0 to 4: (0=0% area; 1=1-25%; 2=26-50%, 3=51-75%,4=76% and above). The mean of all scored glomeruli was presented.Moreover, the extent of global and segmental glomerulosclerosis wasevaluated in the same glomeruli, where <80% sclerosis was referred to assegmental and >80% was referred to as global.

Glomerular area. The glomerular area of mostly 100 randomly selectedglomeruli at a magnification of ×100 was quantitated by counting squarescovered by glomeruli area using a grid and the mean glomeruli area wascalculated.

Immunohistochemistry

Renal tissues were fixed in 4% formaldehyde and embedded in paraffin.The paraffin-embedded tissues were then cut to form tissue slides of 4μm. Immunohistochemistry of the paraffin-embedded tissue slides wasanalyzed using antibodies in the following concentration: monoclonalmouse anti rat CD-68 (ED-1, Serotec MCA341) 1:25. For quantitation ofinterstitial CD68+ staining, the number of positive cells was counted in20 randomly selected non-overlapping fields per animal, and the meanvalue was presented.

Real-time PCR

Kidney RNA was extracted with an RNeasy Fibrous Tissue Mini kit (Qiagen)and after DNAse I treatment, single-stranded cDNA was synthesized from 2μg total RNA using the qScript cDNA Synthesis Kit (Quanta Biosciences)and diluted for real-time PCR. The expression of collagen 4α,fibronectin and TGFβ was quantified using the 7300 Real Time PCR System(Applied Biosystems). The assay was performed according to manufacturerinstructions using the primers (Assay ID) represented at the table belowsupplied by Applied Biosystems. Data were normalized to the referencegene TATA box Binding Protein (TBP) and presented as relative mRNAlevels compared with Sham PBS 0.5% Eth treatment (Table 1).

TABLE 1 Gene Expression references Assay ID Gene Symbol Gene NameRn01482927_m1 Col IVα1 Collagen; type IV; alpha 1 Rn00572010_m1 TGFβ1Transforming growth factor; beta 1 Rn00569575_m1 Fn1 Fibronectin 1Rn01455646_m1 TBP TATA box binding proteinStatistics

Data are expressed as means±SEM. Statistical significance was determinedby one-way ANOVA or Student's t-test where appropriate. Statisticalanalyses were performed using Sigma Stat software.

Results

VB-703 Treatment Effect on Physiological Parameters

VB-703 treatment significantly improved urinary albumin/creatinine/dayratio at the termination of the study (FIG. 22).

VB-703 Treatment Effect on Glomerular Damage

Glomeruli were evaluated for their fibrosis extent by scoring and bycalculation of the percent of glomeruli having segmental sclerosis,global sclerosis and the sum of global and segmental scleroticglomeruli. Moreover, the area of the glomeruli was calculated and thepercent of hypertrophied glomeruli was calculated. Damaged glomeruliincluded hypertrophied (at least ×1.5 from normal area) and or scleroticglomeruli.

VB-703 and telmisartan treatment significantly reduced the damagedglomeruli percent by 38% (p≤0.005) and 31% (p≤0.005) respectively (FIG.23). This effect was partially contributed by the reduction in glomerulihypertrophy. The major contribution to the reduction in glomeruli damagewas due to the reduction in sclerotic glomeruli. VB-703 and telmisartantreatment resulted in a 49% (p≤0.005) and 57% (p≤0.005) reduction ofsclerotic glomeruli, respectively (FIG. 24, Table 2).

TABLE 2 Effect of VB-201 on Glomerular sclerosis (Mean ± S.E)* HealthyPBS Nx Nx Nx 0.5% Sham PBS 0.5% VB-703 Telmisartan Treatment Eth PBS0.5% Eth Eth 4 mg/kg 10 mg/kg Glomerular sclerosis Segmental % 1.0 ±0.58 1.3 ± 0.88 41.0 ± 4.81 22.9 ± 2.83 19.1 ± 4.30 (n = 3) (n = 3) (n =7) (n = 7) (n = 8) p ≤ 0.001 p ≤ 0.001 P < 0.01 P = 0.005 Global % 0.0 ±0.00 0.0 ± 0.00  7.1 ± 4.39  1.9 ± 1.55  1.9 ± 1.60 (n = 3) (n = 3) (n =7) (n = 7) (n = 8) n.s n.s n.s n.s Global & 1.0 ± 0.58 1.3 ± 0.88 48.3 ±5.38 24.7 ± 3.77 21.0 ± 5.45 Segmental % (n = 3) (n = 3) (n = 7) (n = 7)(n = 8) p ≤ 0.001 p ≤ 0.001 P < 0.005 P < 0.005 *Number of animalstested per group and p value versus Nx PBS 0.5% Eth group is presented.

FIG. 25 shows typical sclerotic changes in glomeruli (PAS staining) ofvehicle treated nephrectomized animals in contrast with healthy or shamoperated animals or with VB-703 treated animals, or telmisartan treatedanimal.

VB-703 Treatment Effect on Pro-fibrotic Markers

The mRNA expression of Collagen IV was increased by 7 or 8 foldrespectively in vehicle treated nephrectomized rats (7.5±1.51) incontrast with healthy (1.1±0.12) or sham operated animals (1.0±0.32).VB-703 treatment significantly (p<0.05) reduced Collagen IV expressionby 51% (3.7±0.52) compared to those observed for Nx PBS 0.5% Ethtreatment. A 41% reduction in Collagen IV expression was observed in thetelmisartan treated nephrectomized rats (4.4±0.23) compared to thoseobserved for Nx PBS 0.5% Eth treatment, with marginal significance(p=0.064) (FIG. 26A).

The mRNA expression of fibronectin was increased by 16 or 13 foldrespectively in vehicle treated nephrectomized rats (12.7±1.01) incontrast with healthy (0.8±0.08) or sham operated animals (1.0±0.31).VB-703 treatment significantly (p<0.005) reduced fibronectin expressionby 47% (6.7±0.98) compared to those observed for Nx PBS 0.5% Ethtreatment. Telmisartan treatment moderately reduced fibronectinexpression by 23% (9.8±2.09); however this reduction was notstatistically significant (FIG. 26B).

The mRNA expression of TGF-β was increased by 10 or 8 fold respectivelyin vehicle treated nephrectomized rats (8.4±0.49) in contrast withhealthy (0.9±0.24) or sham operated animals (1.0±0.23) (p≤0.001). VB-703and telmisartan treatment significantly (p≤0.001) reduced TGF-βexpression by 42% (4.8±0.32), and by 44% (4.7±0.52), respectively,compared to those observed for Nx PBS 0.5% Eth treatment (FIG. 26C).

VB-703 Treatment Effect on Glomerular and InterstitialMonocyte/Macrophage Infiltration

FIGS. 27A-27B show the effect of VB-703 on monocyte/macrophage cellinfiltration in the glomeruli (FIG. 27A) or in the interstitium (FIG.27B). In this experiment, VB-703 and telmisartan did not produce asignificant effect on reducing glomerular and interstitialmonocyte/macrophage infiltration.

All publications, patents and patent applications mentioned in thisapplication are herein incorporated in their entirety by reference intothe specification, to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting.

What is claimed is:
 1. A method of treating fibrosis or an inflammatorydisease or disorder mediated by TLR or monocyte migration, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound having a structure according to Formula 1,

or a stereoisomer, a stereoisomeric mixture, or a salt thereof; whereineach of B_(l), B₂, and B₃ is independently selected from the groupconsisting of oxygen, sulfur, nitrogen, phosphorus, and silicon; whereineach of said nitrogen, phosphorus, and silicon is optionally substitutedby one or more substituents selected from the group consisting of alkyl,halo, cycloalkyl, aryl, hydroxy, thiohydroxy, alkoxy, aryloxy,thioaryloxy, thioalkoxy, and oxo; wherein R¹⁰ is a C₂₋₂₈ alkyloptionally substituted by one to five R¹¹substituents, wherein each R¹¹is independently selected from the group consisting of alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heteroaryl, halo, trihalomethyl, hydroxy,alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, phosphonate,phosphate, phosphinyl, sulfonyl, sulfinyl, sulfonamide, amide, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, C-carbamate, N-carbamate,C-thiocarboxy, S-thiocarboxy, and amino; wherein p is an integerselected from 1-10; wherein q is an integer selected from 1-26; whereinR²⁰ is selected from the group consisting of hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, aryl, and heteroaryl; and wherein Het is anoptionally substituted heteroalicyclic ring or an optionally substitutedheteroaryl.
 2. The method of claim 1, wherein the compound has astructure according to Formula 2,

wherein Het is a pyridine and wherein the nitrogen atom of the pyridineis directly connected to the alkylene chain —(CH₂)_(p)—; wherein thepyridine is unsubstituted or substituted by one to five R¹²substituents; and wherein R¹² is a halogen, a C₆₋₁₀ aryl, a heteroaryl,or an alkyl.
 3. The method of claim 2, wherein the compound has astructure according to Formula 3,


4. The method of claim 3, wherein R¹⁰is selected from the groupconsisting of hexadecyl, eicosanyl, and (2′-octyl) dodecyl.
 5. Themethod of claim 3, wherein R²⁰is a hydrogen or a C₁₋₄ alkyl.
 6. Themethod of claim 3, wherein q is an integer of 2-6.
 7. The method ofclaim 3, wherein p is an integer of 2-5.
 8. The method of claim 2,wherein the compound has a structure according to Formula 4,


9. The method of claim 8, wherein the compound has a structure accordingto Formula 5,


10. The method of claim 9, wherein R¹⁰ is selected from the groupconsisting of hexadecyl, eicosanyl, and (2′-octyl) dodecyl, and R²⁰ is ahydrogen or a methyl.
 11. The method of claim 1, wherein the compoundhas a structure selected from the group consisting of:


12. The method of claim 11, wherein the compound is selected from thegroup consisting of(R)-1-hexadecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-701);(R)-1-eicosanyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-702);(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-703);(R)-1-hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-704); and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoricacid pyridiniumethyl ester (VB-705).
 13. The method of claim 12, whereinthe compound is(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-sn-glycero-3-phosphoric acidpyridiniumethyl ester (VB-703).
 14. The method of claim 1, wherein thecompound has a structure selected from the group consisting of:


15. The method of claim 14, wherein the compound is selected from thegroup consisting of(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-fluoro-pyridiniumethyl ester (VB-706) and(R)-1-(2′-octyl)dodecyl-2-(4′-carboxy)butyl-glycero-sn-3-phosphoric acid3-phenyl-pyridiniumethyl ester (VB-707).
 16. The method of claim 1,wherein the method is for treating fibrosis.
 17. The method of claim 16,wherein the fibrosis is pulmonary fibrosis, liver fibrosis, skinfibrosis, kidney fibrosis, idiopathic pulmonary fibrosis, cysticfibrosis, progressive massive fibrosis, cirrhosis, steatohepatitis(fatty liver disease), nonalcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), endomyocardial fibrosis, myocardialinfarction, atrial fibrosis, medastinal fibrosis, myelofibrosis,retroperitoneal fibrosis, nephrogenic systemic fibrosis, keloid, Crohn'sdisease, scleroderma/systemic sclerosis, arthrofibrosis, Peyronie'sdisease, Dupuytren's contracture, adhesive capsulitis, or focal andsegmental glomerulosclerosis.
 18. The method of claim 17, wherein thefibrosis is liver fibrosis.
 19. The method of claim 17, wherein thefibrosis is kidney fibrosis.
 20. The method of claim 1, wherein themethod is for treating an inflammatory disease or disorder mediated byTLR or monocyte migration.
 21. The method of claim 20, wherein theinflammatory disease or disorder is atherosclerosis, rheumatoidarthritis, inflammatory bowel disease, ulcerative colitis, multiplesclerosis, or psoriasis.
 22. The method of claim 20, wherein theinflammatory disease or disorder is an inflammatory cardiovasculardisease or disorder, a cerebrovascular disease or disorder, or aperipheral vascular disease or disorder.
 23. The method of claim 22,wherein the inflammatory disease or disorder is an inflammatorycardiovascular disease or disorder.
 24. The method of claim 23, whereinthe inflammatory cardiovascular disease or disorder is selected from thegroup consisting of occlusive diseases or disorders, atherosclerosis,cardiac valvular disease, stenosis, restenosis, in-stent-stenosis,myocardial infarction, coronary arterial disease, acute coronarysyndromes, congestive heart failure, angina pectoris, myocardialischemia, thrombosis, Wegener's granulomatosis, Takayasu's arteritis,Kawasaki syndrome, anti-factor VIII autoimmune diseases or disorders,necrotizing small vessel vasculitis, microscopic polyangiitis, Churg andStrauss syndrome, pauci-immune focal necrotizing glomerulonephritis,crescentic glomerulonephritis, antiphospholipid syndrome, antibodyinduced heart failure, thrombocytopenic purpura, autoimmune hemolyticanemia, cardiac autoimmunity, Chagas' disease or disorder, andanti-helper T lymphocyte autoimmunity.
 25. The method of claim 22,wherein the cerebrovascular disease or disorder is selected from thegroup consisting of stroke, cerebrovascular inflammation, cerebralhemorrhage, and vertebral arterial insufficiency.
 26. The method ofclaim 22, wherein the peripheral vascular disease or disorder isselected from the group consisting of gangrene, diabetic vasculopathy,ischemic bowel disease, thrombosis, diabetic retinopathy, and diabeticnephropathy.
 27. The method of claim 1, wherein the subject is a human.